Endolysin variant

ABSTRACT

The present invention relates to the field of antimicrobial enzymes. In particular, the present invention relates to a polypeptide comprising an amino acid sequence exhibiting at least 90% sequence identity with the sequence of SEQ ID NO:1, with the proviso that the polypeptide does neither comprise the sequence according to SEQ ID NO:2, nor the sequence according to SEQ ID NO:3, nor the sequence according to SEQ ID NO:4. The present invention relates also to nucleic acids encoding an inventive polypeptide, vectors or bacteriophages comprising an inventive nucleic acid as well as host cells comprising an inventive polypeptide, nucleic acid, vector, and/or bacteriophage. Similarly, the present invention relates to compositions comprising a polypeptide, nucleic acid, vector, bacteriophage, and/or host cell according to the present invention.

This application is a divisional of U.S. application Ser. No.16/304,355, filed Nov. 26, 2018, as a national phase application under35 U.S.C. § 371 of International Application No. PCT/IB2017/053099,filed May 26, 2017, which claims benefit of priority to InternationalApplication No. PCT/TH2016/000048, filed May 27, 2016, the entirecontents of each of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION I. Field of the Invention

The present invention relates to the field of antimicrobial enzymes. Inparticular, the present invention relates to a polypeptide comprising anamino acid sequence exhibiting at least 90% sequence identity with thesequence of SEQ ID NO:1, with the proviso that the polypeptide doesneither comprise the sequence according to SEQ ID NO:2, nor the sequenceaccording to SEQ ID NO:3, nor the sequence according to SEQ ID NO:4. Thepresent invention relates also to nucleic acids encoding an inventivepolypeptide, vectors or bacteriophages comprising an inventive nucleicacid as well as host cells comprising an inventive polypeptide, nucleicacid, vector, and/or bacteriophage. Similarly, the present inventionrelates to compositions comprising a polypeptide, nucleic acid, vector,bacteriophage, and/or host cell according to the present invention.

II. Description of Related Art

Endolysins are peptidoglycan hydrolases encoded by bacteriophages (orbacterial viruses). They are synthesized during late gene expression inthe lytic cycle of phage multiplication and mediate the release ofprogeny virions from infected cells through degradation of the bacterialpeptidoglycan. In terms of enzymatic activity they are usually eitherß(1,4)-glycosylases (lysozymes), transglycosylases, amidases orendopeptidases. Antimicrobial application of endolysins was alreadysuggested in 1991 by Gasson (GB2243611). Although the killing capacityof endolysins has been known for a long time, the use of these enzymesas antibacterials was ignored due to the success and dominance ofantibiotics. Only after the appearance of multiple antibiotic resistantbacteria this simple concept of combating human pathogens withendolysins received interest. A compelling need to develop totally newclasses of antibacterial agents emerged and endolysins used as‘enzybiotics’—a hybrid term of ‘enzymes’ and ‘antibiotics’—perfectly metthis need. In 2001, Fischetti and coworkers demonstrated for the firsttime the therapeutic potential of bacteriophage Cl endolysin towardsgroup A streptococci (Nelson et al., 2001). Since then many publicationshave established endolysins as an attractive and complementaryalternative to control bacterial infections, particularly by Grampositive bacteria. Subsequently different endolysins against other Grampositive pathogens such as Streptococcus pneumoniae (Loeffler et al.,2001), Bacillus anthracis (Schuch et al., 2002), S. agalactiae (Cheng etal., 2005) and Staphylococcus aureus (Rashel et al, 2007) have proventheir efficacy as enzybiotics. Nowadays, the most important challenge ofendolysin therapy lies in the insensitivity of Gram-negative bacteriatowards the exogenous action of endolysins, since the outer membraneshields the access of endolysins from the peptidoglycan. In 2014,Oliveira et al. (PLoS One, 2014 Oct. 7; 9(10):e108376) published areport about a thermostable Salmonella phage endolysin, Lys68, withbroad bactericidal properties against Gram-negative pathogens inpresence of weak acids.

Gram-negative bacteria possess an outer membrane, with itscharacteristic asymmetric bilayer as a hallmark. The outer membranebilayer consists of an inner monolayer containing phospholipids(primarily phosphatidyl ethanolamine) and an outer monolayer that ismainly composed of a single glycolipid, lipopolysaccharide (LPS). Thereis an immense diversity of LPS structures in the bacterial kingdom andthe LPS structure may be modified in response to prevailingenvironmental conditions. The stability of the LPS layer and interactionbetween different LPS molecules is mainly achieved by the electrostaticinteraction of divalent ions (Mg2+, Ca2+) with the anionic components ofthe LPS molecule (phosphate groups in the lipid A and the inner core andcarboxyl groups of KDO). Furthermore, the dense and ordered packing ofthe hydrophobic moiety of lipid A, favored by the absence of unsaturatedfatty acids, forms a rigid structure with high viscosity. This makes itless permeable for lipophilic molecules and confers additional stabilityto the outer membrane (OM).

In contrast to Gram-negative bacteria, Gram-positive bacteria do notpossess an outer membrane. The cytoplasmic membrane is surrounded by anup to 25 nm thick layer of peptidoglycan (which is only up to 5 nm forGram-negative bacteria) which forms the cell wall. Main purpose of thecell wall of Gram-positives is to maintain bacterial shape and tocounteract the internal bacterial cell pressure. Peptidoglycan, ormurein, is a polymer consisting of sugars and amino acids. The sugarcomponent consists of alternating residues of β-(1,4) linkedN-acetylglucosamine and N-acetylmuramic acid residues compose the sugarcomponents. A peptide chain of three to five amino acids is attached tothe N-acetylmuramic acid. The peptide chain can be cross-linked to thepeptide chain of another strand forming a 3D mesh-like layer. Thepeptide chain may contain D- and L-amino acid residues and thecomposition may vary for different bacteria.

Meanwhile, new strategies have emerged to utilize also endolysinsoriginating from phages infecting Gram-negative bacterial species tocontrol infections caused by Gram-negative bacteria. For this purpose,endolysins of Gram negative bacteria are fused with, e.g. cationic,amphipathic, hydrophobic or antimicrobial peptides. This type of fusionprotein allows overcoming previous problems with the outer membrane ofGram-negative bacteria.

However, despite the advances in the art regarding antibacterial agents,there is still a need in the art for further improvement in the designof such antibacterial agents, in particular due to the increasingresistance to conventional antibiotics.

This problem is solved by the subject-matter as set forth below and inthe appended claims.

SUMMARY OF THE INVENTION

In a first aspect the present invention relates to a polypeptidecomprising an amino acid sequence exhibiting at least 90%, preferably atleast 95%, more preferably at least 97% sequence identity with thesequence of SEQ ID NO:1, wherein SEQ ID NO:1 is characterized by

-   -   X5 may be any amino acid, preferably I or V,    -   X13 may be any amino acid, preferably G or S,    -   X72 may be any amino acid, preferably P, L or S,    -   X98 may be any amino acid, preferably G or D,        with the proviso that the polypeptide does neither comprise the        sequence according to SEQ ID NO:2, nor the sequence according to        SEQ ID NO:3, nor the sequence according to SEQ ID NO:4.

The inventor of the present invention has surprisingly found thatremoval of N-terminal and in particular C-terminal portions of theenzyme Lys68 according to SEQ ID NO:2 still yields active enzyme.Moreover, a significant number of mutations can be introduced withoutloss of activity. In parallel, such mutations can be suited to increasethermal stability, thereby making the enzyme more apt for industrialuse.

In a preferred embodiment, the inventive polypeptide comprises an aminoacid sequence exhibiting at least 86% sequence identity with thesequence of SEQ ID NO: 5, wherein SEQ ID NO: 5 is characterized by

-   -   X1 may be absent or any amino acid, in particular M,    -   X11 may be any amino acid, preferably I or V,    -   X19 may be any amino acid, preferably G or S,    -   X78 may be any amino acid, preferably P, L or S,    -   X104 may be any amino acid, preferably G or D;        with the proviso that the polypeptide does neither comprise the        sequence according to SEQ ID NO:2, nor the sequence according to        SEQ ID NO:3, nor the sequence according to SEQ ID NO:4.

In another preferred embodiment the inventive polypeptide comprises anamino acid sequence exhibiting at least 80% sequence identity with thesequence of SEQ ID NO: 6, wherein SEQ ID NO: 6 is characterized by

-   -   X1 may be absent or any amino acid, in particular M,    -   X11 may be any amino acid, preferably I or V,    -   X19 may be any amino acid, preferably G or S,    -   X78 may be any amino acid, preferably P, L or S,    -   X104 may be any amino acid, preferably G or D,    -   X134 may be any amino acid, preferably G or C;        with the proviso that the polypeptide does neither comprise the        sequence according to SEQ ID NO:2, nor the sequence according to        SEQ ID NO:3, nor the sequence according to SEQ ID NO:4.

In a further preferred embodiment, the polypeptide according to thepresent invention comprises an amino acid sequence exhibiting at leastabout 80% sequence identity with the sequence of SEQ ID NO:7, whereinSEQ ID NO:7 is characterized by

-   -   X5 may be any amino acid, preferably I or V,    -   X13 may be any amino acid, preferably G or S,    -   X72 may be any amino acid, preferably P, L or S,    -   X98 may be any amino acid, preferably G or D,    -   X128 may be any amino acid, preferably G or C,        with the proviso that the polypeptide does neither comprise the        sequence according to SEQ ID NO:2, nor the sequence according to        SEQ ID NO:3, nor the sequence according to SEQ ID NO:4.

In a further preferred embodiment, the polypeptide according to thepresent invention comprises an amino acid sequence exhibiting at leastabout 80% sequence identity with the sequence of SEQ ID NO:8, whereinSEQ ID NO:8 is characterized by

-   -   X5 may be any amino acid, preferably I or V,    -   X13 may be any amino acid, preferably G or S,    -   X72 may be any amino acid, preferably P, L or S,    -   X98 may be any amino acid, preferably G or D,    -   X128 may be any amino acid, preferably G or C; and    -   X150 may be any amino acid, preferably A or V;        with the proviso that the polypeptide does neither comprise the        sequence according to SEQ ID NO:2, nor the sequence according to        SEQ ID NO:3, nor the sequence according to SEQ ID NO:4.

In a particularly preferred embodiment, the polypeptide according to thepresent invention comprises an amino acid sequence exhibiting at leastabout 80% sequence identity with the sequence of SEQ ID NO: 9, whereinSEQ ID NO: 9 is characterized by

-   -   X1 may be absent or any amino acid, in particular M,    -   X11 may be any amino acid, preferably I or V,    -   X19 may be any amino acid, preferably G or S,    -   X78 may be any amino acid, preferably P, L or S,    -   X104 may be any amino acid, preferably G or D,    -   X134 may be any amino acid, preferably G or C; and    -   X156 may be any amino acid, preferably A or V;        with the proviso that the polypeptide does neither comprise the        sequence according to SEQ ID NO:2, nor the sequence according to        SEQ ID NO:3, nor the sequence according to SEQ ID NO:4.

In a further aspect, the present invention relates to a polypeptidecomprising an amino acid sequence exhibiting at least 90% sequenceidentity with the sequence of SEQ ID NO: 10, wherein SEQ ID NO: 10 ischaracterized by

-   -   X2 may be any amino acid, preferably D or N,    -   X5 may be any amino acid, preferably L, I or V,    -   X6 may be any amino acid, preferably H or K,    -   X13 may be any amino acid, preferably G, V or S,    -   X15 may be any amino acid, preferably R or Q,    -   X20 may be any amino acid, preferably K or R,    -   X23 may be any amino acid, preferably K or P,    -   X24 may be any amino acid, preferably S or N,    -   X28 may be any amino acid, preferably L or F,    -   X32 may be any amino acid, preferably Y or F,    -   X34 may be any amino acid, preferably H or S,    -   X37 may be any amino acid, preferably A or P,    -   X38 may be any amino acid, preferably D or H,    -   X40 may be any amino acid, preferably K or Y,    -   X49 may be any amino acid, preferably Q or R,    -   X55 may be any amino acid, preferably H or N,    -   X56 may be any amino acid, preferably K or R,    -   X59 may be any amino acid, preferably V, S or A,    -   X72 may be any amino acid, preferably P, L, T or S,    -   X81 may be any amino acid, preferably M or V,    -   X90 may be any amino acid, preferably V, P or A,    -   X95 may be any amino acid, preferably A or V,    -   X98 may be any amino acid, preferably G or D,    -   X108 may be any amino acid, preferably V, I or A,    -   X109 may be any amino acid, preferably A or S,    -   X113 may be any amino acid, preferably N or S,    -   X116 may be any amino acid, preferably S or T;        with the proviso that the polypeptide does neither comprise the        sequence according to SEQ ID NO:2, nor the sequence according to        SEQ ID NO:3, nor the sequence according to SEQ ID NO:4, nor        consists of any of the following sequences: SEQ ID NO:11, SEQ ID        NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,        SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID        NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,        SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID        NO:30, and SEQ ID NO:31.

The inventive polypeptide may comprise additional amino acid sequenceelements. For example the inventive polypeptide may additionallycomprise at least one amino acid sequence selected from the groupconsisting of amphipathic peptides, cationic peptides, hydrophobicpeptides, naturally occurring antimicrobial peptides, sushi peptides anddefensins. Such further peptide can enhance the antibacterial activityof the inventive polypeptide.

In further aspects, the present invention relates to nucleic acidsencoding an inventive polypeptide, vectors or bacteriophages comprisingan inventive nucleic acid as well as host cells comprising an inventivepolypeptide, nucleic acid, vector, and/or bacteriophage.

Finally, the present invention relates in a further aspect also tocompositions comprising a polypeptide, nucleic acid, vector,bacteriophage, and/or host cell according to the present invention.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. Definitions

The term “polypeptide” as used herein refers in particular to a polymerof amino acid residues linked by peptide bonds in a specific sequence.The amino acid residues of a polypeptide may be modified by e.g.covalent attachments of various groups such as carbohydrates andphosphate. Other substances may be more loosely associated with thepolypeptide, such as heme or lipid, giving rise to conjugatedpolypeptides which are also comprised by the term “polypeptide” as usedherein. The term as used herein is intended to encompass also proteins.Thus, the term “polypeptide” also encompasses for example complexes oftwo or more amino acid polymer chains. The term “polypeptide” doesencompass embodiments of polypeptides which exhibit optionallymodifications typically used in the art, e.g. biotinylation,acetylation, pegylation, chemical changes of the amino-, SH- orcarboxyl-groups (e.g. protecting groups) etc. As will become apparentfrom the description below, the polypeptide according to the inventionmay be an artificially engineered polypeptide, which does not exist inthis form in nature. Such polypeptide may for example exhibit artificialmutations vis-à-vis a naturally occurring polypeptide or may compriseheterologous sequences, or may be a fragment of a naturally occurringpolypeptide, which fragment does not occur in this form in nature.Furthermore, the polypeptide according to the present invention may be afusion protein, i.e. represent the linkage of at least two amino acidsequences which do not occur in this combination in nature. The term“polypeptide” as used herein is not limited to a specific length of theamino acid polymer chain, but typically the polypeptide will exhibit alength of more than about 125 amino acids. Usually, but not necessarily,a typical polypeptide of the present invention will not exceed about1000 amino acids in length. The inventive polypeptide may for instancebe at most about 750 amino acids long, at most about 500 amino acidslong or at most about 300 amino acids long. A possible length range forthe inventive polypeptide, without being limited thereto, may thus forexample be about 200 to about 750 amino acids, or about 225 to about 600amino acids, or about 250 to about 350 amino acids.

The term “% sequence identity” is generally understood in the art. Twosequences to be compared are aligned to give a maximum correlationbetween the sequences. This may include inserting “gaps” in either oneor both sequences, to enhance the degree of alignment. A % identity maythen be determined over the whole length of each of the sequences beingcompared (so-called global alignment), that is particularly suitable forsequences of the same or similar length, or over shorter, definedlengths (so-called local alignment), that is more suitable for sequencesof unequal length. In the above context, an amino acid sequence having a“sequence identity” of at least, for example, 95% to a query amino acidsequence, is intended to mean that the sequence of the subject aminoacid sequence is identical to the query sequence except that the subjectamino acid sequence may include up to five amino acid alterations pereach 100 amino acids of the query amino acid sequence. In other words,to obtain an amino acid sequence having a sequence of at least 95%identity to a query amino acid sequence, up to 5% (5 of 100) of theamino acid residues in the subject sequence may be inserted orsubstituted with another amino acid or deleted. Methods for comparingthe identity and homology of two or more sequences are well known in theart. The percentage to which two sequences are identical can for examplebe determined by using a mathematical algorithm. A preferred, but notlimiting, example of a mathematical algorithm which can be used is thealgorithm of Karlin et al. (1993), PNAS USA, 90:5873-5877. Such analgorithm is integrated in the BLAST family of programs, e.g. BLAST orNBLAST program (see also Altschul et al., 1990, J. Mol. Biol. 215,403-410 or Altschul et al. (1 997), Nucleic Acids Res, 25:3389-3402),accessible through the home page of the NCBI at world wide web sitencbi.nlm.nih.gov) and FASTA (Pearson (1990), Methods Enzymol. 83, 63-98;Pearson and Lipman (1988), Proc. Natl. Acad. Sci. U. S. A 85,2444-2448.). Sequences which are identical to other sequences to acertain extent can be identified by these programs. Furthermore,programs available in the Wisconsin Sequence Analysis Package, version9.1 (Devereux et al, 1984, Nucleic Acids Res., 387-395), for example theprograms BESTFIT and GAP, may be used to determine the % identitybetween two polypeptide sequences. BESTFIT uses the “local homology”algorithm of (Smith and Waterman (1981), J. Mol. Biol. 147, 195-197.)and finds the best single region of similarity between two sequences. Ifherein reference is made to an amino acid sequence sharing a particulardegree of sequence identity to a reference sequence, then saiddifference in sequence is preferably due to conservative amino acidsubstitutions. Preferably, such sequence retains the activity of thereference sequence, albeit maybe at a slower (or even faster) rate. Inaddition, if reference is made herein to a sequence sharing “at least”at certain percentage of sequence identity, then 100% sequence identityare preferably not encompassed.

As used herein, the term “cationic peptide” refers preferably to apeptide having positively charged amino acid residues. Preferably acationic peptide has a pKa-value of 9.0 or greater. Typically, at leastfour of the amino acid residues of the cationic peptide can bepositively charged, for example, lysine or arginine. “Positivelycharged” refers to the side chains of the amino acid residues which havea net positive charge at about physiological conditions. The term“cationic peptide” as used herein refers also to polycationic peptides,but also includes cationic peptides which comprise for example less than20%, preferably less than 10% positively charged amino acid residues.

The term “polycationic peptide”, as used herein, refers preferably to apeptide composed of mostly positively charged amino acid residues, inparticular lysine and/or arginine residues. A peptide is composed ofmostly positively charged amino acid residues if at least about 20, 30,40, 50, 60, 70, 75, 80, 85, 90, 95 or about 100% of the amino acidresidues are positively charged amino acid residues, in particularlysine and/or arginine residues. The amino acid residues being notpositively charged amino acid residues can be neutrally charged aminoacid residues and/or negatively charged amino acid residues and/orhydrophobic amino acid residues. Preferably the amino acid residuesbeing not positively charged amino acid residues are neutrally chargedamino acid residues, in particular serine and/or glycine.

The term, “antimicrobial peptide” (AMP), as used herein, referspreferably to any naturally occurring peptide that has microbicidaland/or microbistatic activity on for example bacteria, viruses, fungi,yeasts, mycoplasma and protozoa. Thus, the term “antimicrobial peptide”as used herein refers in particular to any peptide havinganti-bacterial, anti-fungal, anti-mycotic, anti-parasitic,anti-protozoal, anti-viral, anti-infectious, anti-infective and/orgermicidal, algicidal, amoebicidal, microbicidal, bactericidal,fungicidal, parasiticidal, protozoacidal, protozoicidal properties.Preferred are anti-bacterial peptides. The antimicrobial peptide may bea member of the RNase A super family, a defensin, cathelicidin,granulysin, histatin, psoriasin, dermicidine or hepcidin. Theantimicrobial peptide may be naturally occurring in insects, fish,plants, arachnids, vertebrates or mammals. Preferably the antimicrobialpeptide may be naturally occurring in insects, fish, plants, arachnids,vertebrates or mammals. Preferably the antimicrobial peptide may benaturally occurring in radish, silk moth, wolf spider, frog, preferablyin Xenopus laevis, Rana frogs, more preferably in Rana catesbeiana,toad, preferably Asian toad Bufo bufo gargarizans, fly, preferably inDrosophila, more preferably in Drosophila melanogaster, in Aedesaegypti, in honey bee, bumblebee, preferably in Bombus pascuorum, fleshfly, preferably in Sarcophaga peregrine, scorpion, horseshoe crab,catfish, preferably in Parasilurus asotus, cow, pig, sheep, porcine,bovine, monkey and human. As used herein, an “antimicrobial peptide”(AMP) may in particular be a peptide which is not a cationic peptide,polycationic peptide, amphipathic peptide, sushi peptide, defensins, andhydrophobic peptide, but nevertheless exhibits antimicrobial activity.

The term “sushi peptide”, as used herein, refers to complement controlproteins (CCP) having short consensus repeats. The sushi module of sushipeptides functions as a protein-protein interaction domain in manydifferent proteins. Peptides containing a Sushi domain have been shownto have antimicrobial activities. Preferably, sushi peptides arenaturally occurring peptides.

The term “amphipathic peptide”, as used herein, refers to syntheticpeptides having both hydrophilic and hydrophobic functional groups.Preferably, the term “amphipathic peptide” as used herein refers to apeptide having a defined arrangement of hydrophilic and hydrophobicgroups e.g. amphipathic peptides may be e.g. alpha helical, havingpredominantly non polar side chains along one side of the helix andpolar residues along the rest of its surface.

The term “hydrophobic group”, as used herein, refers preferably tochemical groups such as amino acid side chains which are substantiallywater insoluble, but soluble in an oil phase, with the solubility in theoil phase being higher than that in water or in an aqueous phase. Inwater, amino acid residues having a hydrophobic side chain interact withone another to generate a non-aqueous environment. Examples of aminoacid residues with hydrophobic side chains are valine, isoleucine,leucine, methionine, phenylalanine, tryptophan, cysteine, alanine,tyrosine, and proline residues.

The term “hydrophobic peptide”, as used herein, refers to a hydrophobicpeptide, which is preferably composed of mostly amino acid residues withhydrophobic groups. Such peptide is preferably composed of mostlyhydrophobic amino acid residues, i.e. at least about 20, 30, 40, 50, 60,70, 75, 80, 85, 90, 95 or at least about 100% of the amino acid residuesare hydrophobic amino acid residues. The amino acid residues being nothydrophobic are preferably neutral and preferably not hydrophilic.

The term “comprising”, as used herein, shall not be construed as beinglimited to the meaning “consisting of” (i.e. excluding the presence ofadditional other matter). Rather, “comprising” implies that optionallyadditional matter may be present. The term “comprising” encompasses asparticularly envisioned embodiments falling within its scope “consistingof” (i.e. excluding the presence of additional other matter) and“comprising but not consisting of” (i.e. requiring the presence ofadditional other matter), with the former being more preferred.

The use of the word “a” or “an”, when used herein, may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

II. Polypeptides

As already mentioned, the present invention relates to a polypeptidecomprising an amino acid sequence exhibiting at least 90%, preferably atleast 95%, more preferably at least 97% sequence identity with thesequence of SEQ ID NO:1, wherein SEQ ID NO:1 is characterized by

-   -   X5 of SEQ ID NO:1 may be any amino acid, preferably I or V,    -   X13 of SEQ ID NO:1 may be any amino acid, preferably G or S,    -   X72 of SEQ ID NO:1 may be any amino acid, preferably P, L or S,    -   X98 of SEQ ID NO:1 may be any amino acid, preferably G or D;        with the proviso that the polypeptide does neither comprise the        sequence according to SEQ ID NO:2, nor the sequence according to        SEQ ID NO:3, nor the sequence according to SEQ ID NO:4.

A person skilled in the art will understand, that when herein referenceis made to a sequence, here for example SEQ ID NO:1, and specificresidues are referred to with “X”, followed by a number, then this isintended to refer to the respective residue position in said sequence.“X5” for example refers to the “X” residue at position 5 of SEQ ID NO:1,counted from the N-terminus of SEQ ID NO:1. Said position need notnecessarily reflect the actual position in the polypeptide, which maycomprise additional sequence elements, e.g. at its N-terminus. The sameapplies of course when the same terminology is used for other sequencesdisclosed herein.

In some embodiments of the invention, at least one of the following fourconditions applies for the inventive polypeptide comprising an aminoacid sequence exhibiting at least 90% sequence identity with thesequence of SEQ ID NO:1:

-   -   i) X5 of SEQ ID NO:1 is I or V, preferably V,    -   ii) X13 of SEQ ID NO:1 is G or S, preferably S,    -   iii) X72 of SEQ ID NO:1 is P, L or S, preferably L or S,        -   more preferably S, and/or    -   iv) X98 of SEQ ID NO:1 is G or D, preferably D.        That “at least one” of these conditions applies includes that,        e.g., one, two, three or all four conditions may apply. In some        embodiments of the invention, all four conditions apply, i.e.        the following applies for the inventive polypeptide comprising        an amino acid sequence exhibiting at least 90% sequence identity        with the sequence of SEQ ID NO:1:    -   X5 of SEQ ID NO:1 is I or V, preferably V,    -   X13 of SEQ ID NO:1 is G or S, preferably S,    -   X72 of SEQ ID NO:1 is P, L or S, preferably L or S,        -   more preferably S, and    -   X98 of SEQ ID NO:1 is G or D, preferably D.

In some embodiments of the invention, at least one of the following fourconditions applies for the inventive polypeptide comprising an aminoacid sequence exhibiting at least 90% sequence identity with thesequence of SEQ ID NO:1:

-   -   i) X5 of SEQ ID NO:1 is not I,    -   ii) X13 of SEQ ID NO:1 is not G,    -   iii) X72 of SEQ ID NO:1 is not P, and/or    -   iv) X98 of SEQ ID NO:1 is not G.        That “at least one” of these conditions applies includes that,        e.g., one, two, three or all four conditions may apply.

In some embodiments of the invention, at least one of the following fourconditions applies for the inventive polypeptide comprising an aminoacid sequence exhibiting at least 90% sequence identity with thesequence of SEQ ID NO:1:

-   -   i) X5 of SEQ ID NO:1 is V,    -   ii) X13 of SEQ ID NO:1 is S,    -   iii) X72 of SEQ ID NO:1 is L or S, preferably S, and/or    -   iv) X98 of SEQ ID NO:1 is D.        As before, that “at least one” of these conditions applies        includes that, e.g., one, two, three or all four conditions may        apply. Hence, in some embodiments of the invention all four        conditions apply, i.e. the following applies for the inventive        polypeptide comprising an amino acid sequence exhibiting at        least 90% sequence identity with the sequence of SEQ ID NO:1:    -   X5 of SEQ ID NO:1 is V,    -   X13 of SEQ ID NO:1 is S,    -   X72 of SEQ ID NO:1 is L or S, preferably S, and    -   X98 of SEQ ID NO:1 is D.        If only one condition applies, it is particularly preferred if        this means that X72 of SEQ ID NO:1 is L or S, preferably S.        Aside of the situations, where only one of these condition        applies (i.e. X5 of SEQ ID NO:1 is V, X13 of SEQ ID NO:1 is S,        X72 of SEQ ID NO:1 is L or S, preferably S, or X98 of SEQ ID        NO:1 is D), particularly preferred combinations are i) wherein        X72 of SEQ ID NO:1 is L and X98 of SEQ ID NO:1 is D, ii) X13 of        SEQ ID NO:1 is S and X72 of SEQ ID NO:1 is S, and/or iii) X5 of        SEQ ID NO:1 is V and X72 of SEQ ID NO:1 is S.

In particularly preferred embodiments of the invention, the polypeptidecomprising an amino acid sequence exhibiting at least 90% sequenceidentity with the sequence of SEQ ID NO:1 does not exhibit theinactivating mutation E18A described in Oliveira et al. (PLoS One, 2014Oct. 7; 9(10):e108376) (see SEQ ID NO:4). While an inactivated enzymemay also have utility in various technical applications (e.g. asmatching negative control), the active enzymes are still more preferredby the inventor. The polypeptide comprising an amino acid sequenceexhibiting at least 90% sequence identity with the sequence of SEQ IDNO:1 does thus preferably not comprise an alanine residue at saidposition, i.e. does not comprise an amino acid sequence which deviates(inter alia) with a E12A mutation from SEQ ID NO:1. More preferably, thepolypeptide comprising an amino acid sequence exhibiting at least 90%sequence identity with the sequence of SEQ ID NO:1 does retain theoriginal glutamate residue (E) at said position, i.e. retains E12 of SEQID NO:1 and does not deviate from SEQ ID NO:1 at said position.

The amino acid sequence exhibiting at least 90% sequence identity withthe sequence of SEQ ID NO:1 may deviate from SEQ ID NO:1 for example atposition 29, i.e. the threonine residue may be replaced by any otheramino acid, e.g. an alanine residue. Preferably, deviations from SEQ IDNO:1 are due to conservative amino acid substitutions.

As mentioned above, the present invention relates to a polypeptidecomprising an amino acid sequence exhibiting at least about 90% sequenceidentity with the sequence of SEQ ID NO:1. In more preferred embodimentssaid amino acid sequence deviates less than 10% from SEQ ID NO:1. Forexample, the polypeptide according to the invention may comprise anamino acid sequence exhibiting at least about 95%, at least about 96%,at least about 97%, at least about 98%, at least about 99%, at leastabout 99.5% or even 100% sequence identity with the sequence of SEQ IDNO:1. In cases where the inventive polypeptide comprises an amino acidsequence exhibiting 100% sequence identity with the sequence of SEQ IDNO:1, the inventive polypeptide comprises the sequence of SEQ ID NO:1.Such embodiment is particularly contemplated by the inventor.

In a further embodiment of the present invention, the polypeptidecomprising an amino acid sequence exhibiting at least about 90% sequenceidentity with the sequence of SEQ ID NO:1 is a polypeptide, whichcomprises an amino acid sequence exhibiting at least about 86% sequenceidentity with the sequence of SEQ ID NO: 5, wherein SEQ ID NO: 5 ischaracterized by

-   -   X1 of SEQ ID NO: 5 may be absent or any amino acid, in        particular M,    -   X11 of SEQ ID NO: 5 may be any amino acid, preferably I or V,    -   X19 of SEQ ID NO: 5 may be any amino acid, preferably G or S,    -   X78 of SEQ ID NO: 5 may be any amino acid, preferably P, L or S,        and    -   X104 of SEQ ID NO: 5 may be any amino acid, preferably G or D.        It is understood that for such polypeptide still the general        proviso of the present application for polypeptides applies,        i.e. that such polypeptide does neither comprise the sequence        according to SEQ ID NO:2, nor the sequence according to SEQ ID        NO:3, nor the sequence according to SEQ ID NO:4.

In some embodiments of the invention, at least one of the following fiveconditions applies for the inventive polypeptide comprising an aminoacid sequence exhibiting at least 86% sequence identity with thesequence of SEQ ID NO: 5:

-   -   i) X1 of SEQ ID NO: 5 is absent or M, preferably M,    -   ii) X11 of SEQ ID NO: 5 is I or V, preferably V,    -   iii) X19 of SEQ ID NO: 5 is G or S, preferably S,    -   iv) X78 of SEQ ID NO: 5 is P, L or S, preferably L or S, and/or        -   more preferably S,    -   v) X104 of SEQ ID NO: 5 is G or D, preferably D.        That “at least one” of these conditions applies includes that,        e.g., one, two, three, four or all five conditions may apply. In        some embodiments of the invention, all five conditions apply,        i.e. the following applies for the inventive polypeptide        comprising an amino acid sequence exhibiting at least 86%        sequence identity with the sequence of SEQ ID NO: 5:    -   i) X1 of SEQ ID NO: 5 is absent or M, preferably M,    -   ii) X11 of SEQ ID NO: 5 is I or V, preferably V,    -   iii) X19 of SEQ ID NO: 5 is G or S, preferably S,    -   iv) X78 of SEQ ID NO: 5 is P, L or S, preferably L or S,        -   more preferably S, and    -   v) X104 of SEQ ID NO: 5 is G or D, preferably D.

In some embodiments of the invention, at least one of the following fiveconditions applies for the inventive polypeptide comprising an aminoacid sequence exhibiting at least 86% sequence identity with thesequence of SEQ ID NO: 5:

-   -   i) X1 of SEQ ID NO: 5 is absent,    -   ii) X11 of SEQ ID NO: 5 is not I,    -   iii) X19 of SEQ ID NO: 5 is not G,    -   iv) X78 of SEQ ID NO: 5 is not P, and/or    -   v) X104 of SEQ ID NO: 5 is not G.        That “at least one” of these conditions applies includes that,        e.g., one, two, three, four or all five conditions may apply.

In some embodiments of the invention, at least one of the following fiveconditions applies for the inventive polypeptide comprising an aminoacid sequence exhibiting at least 86% sequence identity with thesequence of SEQ ID NO: 5:

-   -   i) X1 of SEQ ID NO: 5 is M,    -   ii) X11 of SEQ ID NO: 5 is V,    -   iii) X19 of SEQ ID NO: 5 is S,    -   iv) X78 of SEQ ID NO: 5 is L or S, preferably S, and/or    -   v) X104 of SEQ ID NO: 5 is D.        As before, that “at least one” of these conditions applies        includes that, e.g., one, two, three, four or all five        conditions may apply. Hence, in some embodiments of the        invention all five conditions apply, i.e. the following applies        for the inventive polypeptide comprising an amino acid sequence        exhibiting at least 86% sequence identity with the sequence of        SEQ ID NO: 5:    -   i) X1 of SEQ ID NO: 5 is M,    -   ii) X11 of SEQ ID NO: 5 is V,    -   iii) X19 of SEQ ID NO: 5 is S,    -   iv) X78 of SEQ ID NO: 5 is L or S, preferably S, and    -   v) X104 of SEQ ID NO: 5 is D.        If only one condition applies, it is particularly preferred if        this means that X78 of SEQ ID NO: 5 is L or S, preferably S.        Aside of the situations, where only one of these condition        applies (i.e. X1 of SEQ ID NO: 5 is M, X11 of SEQ ID NO: 5 is V,        X19 of SEQ ID NO: 5 is S, X78 of SEQ ID NO: 5 is L or S,        preferably S, or X104 of SEQ ID NO: 5 is D), particularly        preferred combinations are wherein at least i) X78 of SEQ ID NO:        5 is L and X104 of SEQ ID NO: 5 is D, ii) X19 of SEQ ID NO: 5 is        S and X78 of SEQ ID NO: 5 is S, and/or iii) X11 of SEQ ID NO: 5        is V and X78 of SEQ ID NO: 5 is S.

If the inventive polypeptide comprises one or more amino acid residuesN-terminal of the amino acid sequence exhibiting at least 86% sequenceidentity with the sequence of SEQ ID NO: 5, then it is preferred that X1of SEQ ID NO: 5 is not M, i.e. absent or any other amino acid. In thealternative constellation, i.e. where the inventive polypeptide does notcomprise one or more amino acid residues N-terminal of the amino acidsequence exhibiting at least 86% sequence identity with the sequence ofSEQ ID NO: 5, it is preferred if X1 of SEQ ID NO: 5 is M, in particularif the polypeptide is to be expressed by recombinant means.

In particularly preferred embodiments of the invention, the polypeptidecomprising an amino acid sequence exhibiting at least 86% sequenceidentity with the sequence of SEQ ID NO: 5 does again not exhibit theinactivating mutation E18A described in Oliveira et al. (PLoS One, 2014Oct. 7; 9(10):e108376) (see SEQ ID NO:4). The polypeptide comprising anamino acid sequence exhibiting at least 86% sequence identity with thesequence of SEQ ID NO: 5 does thus preferably not comprise an alanineresidue at said position, i.e. does not comprise an amino acid sequencewhich deviates (inter alia) with a E18A mutation from SEQ ID NO: 5. Morepreferably, the polypeptide comprising an amino acid sequence exhibitingat least 86% sequence identity with the sequence of SEQ ID NO: 5 doesretain the original glutamate residue (E) at said position, i.e. retainsE18 of SEQ ID NO: 5 and does not deviate from SEQ ID NO: 5 at saidposition.

The amino acid sequence exhibiting at least 86% sequence identity withthe sequence of SEQ ID NO: 5 may deviate from SEQ ID NO: 5 for exampleat position 35, i.e. the threonine residue may be replaced by any otheramino acid, e.g. an alanine residue. Another preferred region in whichthe polypeptide may deviate from the sequence of SEQ ID NO: 5 are forexample residues 1 to 6 of SEQ ID NO: 5. Preferably, deviations from SEQID NO: 5 are due to conservative amino acid substitutions.

As mentioned above, the present invention relates to a polypeptidecomprising an amino acid sequence exhibiting at least about 86% sequenceidentity with the sequence of SEQ ID NO: 5. In more preferredembodiments said amino acid sequence deviates less than 14% from SEQ IDNO: 5. For example, the polypeptide according to the invention maycomprise an amino acid sequence exhibiting at least about 90%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,at least about 99%, at least about 99.5% or even 100% sequence identitywith the sequence of SEQ ID NO: 5. In cases where the inventivepolypeptide comprises an amino acid sequence exhibiting 100% sequenceidentity with the sequence of SEQ ID NO: 5, the inventive polypeptidecomprises the sequence of SEQ ID NO: 5. Such embodiment is particularlycontemplated by the inventor.

In a further embodiment of the present invention, the polypeptidecomprising an amino acid sequence exhibiting at least about 90% sequenceidentity with the sequence of SEQ ID NO:1 is a polypeptide, whichcomprises an amino acid sequence exhibiting at least about 80% sequenceidentity with the sequence of SEQ ID NO: 6, wherein SEQ ID NO: 6 ischaracterized by

-   -   X1 of SEQ ID NO: 6 may be absent or any amino acid, in        particular M,    -   X11 of SEQ ID NO: 6 may be any amino acid, preferably I or V,    -   X19 of SEQ ID NO: 6 may be any amino acid, preferably G or S,    -   X78 of SEQ ID NO: 6 may be any amino acid, preferably P, L or S,    -   X104 of SEQ ID NO: 6 may be any amino acid, preferably G or D,        and    -   X134 of SEQ ID NO: 6 may be any amino acid, preferably G or C.        It is again understood that for such polypeptide still the        general proviso of the present application for polypeptides        applies, i.e. that such polypeptide does neither comprise the        sequence according to SEQ ID NO:2, nor the sequence according to        SEQ ID NO:3, nor the sequence according to SEQ ID NO:4.

In some embodiments of the invention, at least one of the following sixconditions applies for the inventive polypeptide comprising an aminoacid sequence exhibiting at least 80% sequence identity with thesequence of SEQ ID NO: 6:

-   -   i) X1 of SEQ ID NO: 6 is absent or M, preferably M,    -   ii) X11 of SEQ ID NO: 6 is I or V, preferably V,    -   iii) X19 of SEQ ID NO: 6 is G or S, preferably S,    -   iv) X78 of SEQ ID NO: 6 is P, L or S, preferably L or S,        -   more preferably S,    -   v) X104 of SEQ ID NO: 6 is G or D, preferably D, and/or    -   vi) X134 of SEQ ID NO: 6 is G or C, preferably C.        That “at least one” of these conditions applies includes that,        e.g., one, two, three, four, five, or all six conditions may        apply. In some embodiments of the invention, all six conditions        apply, i.e. the following applies for the inventive polypeptide        comprising an amino acid sequence exhibiting at least 80%        sequence identity with the sequence of SEQ ID NO: 6:    -   i) X1 of SEQ ID NO: 6 is absent or M, preferably M,    -   ii) X11 of SEQ ID NO: 6 is I or V, preferably V,    -   iii) X19 of SEQ ID NO: 6 is G or S, preferably S,    -   iv) X78 of SEQ ID NO: 6 is P, L or S, preferably L or S,        -   more preferably S,    -   v) X104 of SEQ ID NO: 6 is G or D, preferably D, and    -   vi) X134 of SEQ ID NO: 6 is G or C, preferably C.

In some embodiments of the invention, at least one of the following sixconditions applies for the inventive polypeptide comprising an aminoacid sequence exhibiting at least 80% sequence identity with thesequence of SEQ ID NO: 6:

-   -   i) X1 of SEQ ID NO: 6 is absent,    -   ii) X11 of SEQ ID NO: 6 is not I,    -   iii) X19 of SEQ ID NO: 6 is not G,    -   iv) X78 of SEQ ID NO: 6 is not P,    -   v) X104 of SEQ ID NO: 6 is not G, and/or    -   vi) X134 of SEQ ID NO: 6 is not G.        That “at least one” of these conditions applies includes that,        e.g., one, two, three, four, five or all six conditions may        apply.

In some embodiments of the invention, at least one of the following sixconditions applies for the inventive polypeptide comprising an aminoacid sequence exhibiting at least 80% sequence identity with thesequence of SEQ ID NO: 6:

-   -   i) X1 of SEQ ID NO: 6 is M,    -   ii) X11 of SEQ ID NO: 6 is V,    -   iii) X19 of SEQ ID NO: 6 is S,    -   iv) X78 of SEQ ID NO: 6 is L or S, preferably S,    -   v) X104 of SEQ ID NO: 6 is D, and/or    -   vi) X134 of SEQ ID NO: 6 is C.        As before, that “at least one” of these conditions applies        includes that, e.g., one, two, three, four, five or all six        conditions may apply. Hence, in some embodiments of the        invention all six conditions apply, i.e. the following applies        for the inventive polypeptide comprising an amino acid sequence        exhibiting at least 80% sequence identity with the sequence of        SEQ ID NO: 6:    -   i) X1 of SEQ ID NO: 6 is M,    -   ii) X11 of SEQ ID NO: 6 is V,    -   iii) X19 of SEQ ID NO: 6 is S,    -   iv) X78 of SEQ ID NO: 6 is L or S, preferably S,    -   v) X104 of SEQ ID NO: 6 is D, and    -   vi) X134 of SEQ ID NO: 6 is C.        If only one condition applies, it is particularly preferred if        this means that X78 of SEQ ID NO: 6 is L or S, preferably S.        Aside of the situations, where only one of these condition        applies (i.e. X1 of SEQ ID NO: 6 is M, X11 of SEQ ID NO: 6 is V,        X19 of SEQ ID NO: 6 is S, X78 of SEQ ID NO: 6 is L or S,        preferably S, X104 of SEQ ID NO: 6 is D or X134 of SEQ ID NO: 6        is C), particularly preferred combinations are wherein at        least i) X78 of SEQ ID NO: 6 is L and X104 of SEQ ID NO: 6 is        D, ii) X19 of SEQ ID NO: 6 is S and X78 of SEQ ID NO: 6 is        S, iii) X11 of SEQ ID NO: 6 is V and X78 of SEQ ID NO: 6 is S,        and/or iv) X78 of SEQ ID NO: 6 is S and X134 of SEQ ID NO: 6 is        C.

If the inventive polypeptide comprises one or more amino acid residuesN-terminal of the amino acid sequence exhibiting at least 80% sequenceidentity with the sequence of SEQ ID NO: 6, then it is preferred that X1of SEQ ID NO: 6 is not M, i.e. absent or any other amino acid. In thealternative constellation, i.e. where the inventive polypeptide does notcomprise one or more amino acid residues N-terminal of the amino acidsequence exhibiting at least 80% sequence identity with the sequence ofSEQ ID NO: 6, it is preferred if X1 of SEQ ID NO: 6 is M, in particularif the polypeptide is to be expressed by recombinant means.

In particularly preferred embodiments of the invention, the polypeptidecomprising an amino acid sequence exhibiting at least 80% sequenceidentity with the sequence of SEQ ID NO: 6 does again not exhibit theinactivating mutation E18A described in Oliveira et al. (PLoS One, 2014Oct. 7; 9(10):e108376) (see SEQ ID NO:4). The polypeptide comprising anamino acid sequence exhibiting at least 80% sequence identity with thesequence of SEQ ID NO: 6 does thus preferably not comprise an alanineresidue at said position, i.e. does not comprise an amino acid sequencewhich deviates (inter alia) with a E18A mutation from SEQ ID NO: 6. Morepreferably, the polypeptide comprising an amino acid sequence exhibitingat least 80% sequence identity with the sequence of SEQ ID NO: 6 doesretain the original glutamate residue (E) at said position, i.e. retainsE18 of SEQ ID NO: 6 and does not deviate from SEQ ID NO: 6 at saidposition.

The amino acid sequence exhibiting at least 80% sequence identity withthe sequence of SEQ ID NO: 6 may deviate from SEQ ID NO: 6 for exampleat position 35, i.e. the threonine residue may be replaced by any otheramino acid, e.g. an alanine residue. Other preferred regions in whichthe polypeptide may deviate from the sequence of SEQ ID NO: 6 are forexample residues 1 to 6 of SEQ ID NO: 6 and/or residues 133 to 148 ofSEQ ID NO: 6. Preferably, deviations from SEQ ID NO: 6 are due toconservative amino acid substitutions.

As mentioned above, the present invention relates to a polypeptidecomprising an amino acid sequence exhibiting at least about 80% sequenceidentity with the sequence of SEQ ID NO: 6. In more preferredembodiments said amino acid sequence deviates less than 20% from SEQ IDNO: 6. For example, the polypeptide according to the invention maycomprise an amino acid sequence exhibiting at least about 85%, at leastabout 90%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, at least about 99%, at least about 99.5% or even100% sequence identity with the sequence of SEQ ID NO: 6. In cases wherethe inventive polypeptide comprises an amino acid sequence exhibiting100% sequence identity with the sequence of SEQ ID NO: 6, the inventivepolypeptide comprises the sequence of SEQ ID NO: 6. Such embodiment isparticularly contemplated by the inventor.

In a further embodiment of the present invention, the polypeptidecomprising an amino acid sequence exhibiting at least about 90% sequenceidentity with the sequence of SEQ ID NO:1 is a polypeptide, whichcomprises an amino acid sequence exhibiting at least about 80% sequenceidentity with the sequence of SEQ ID NO:7, wherein SEQ ID NO:7 ischaracterized by

-   -   X5 of SEQ ID NO:7 may be any amino acid, preferably I or V,    -   X13 of SEQ ID NO:7 may be any amino acid, preferably G or S,    -   X72 of SEQ ID NO:7 may be any amino acid, preferably P, L or S,    -   X98 of SEQ ID NO:7 may be any amino acid, preferably G or D, and    -   X128 of SEQ ID NO:7 may be any amino acid, preferably G or C.        It is again understood that for such polypeptide still the        general proviso of the present application for polypeptides        applies, i.e. that such polypeptide does neither comprise the        sequence according to SEQ ID NO:2, nor the sequence according to        SEQ ID NO:3, nor the sequence according to SEQ ID NO:4.

In some embodiments of the invention, at least one of the following fiveconditions applies for the inventive polypeptide comprising an aminoacid sequence exhibiting at least 80% sequence identity with thesequence of SEQ ID NO:7:

-   -   i) X5 of SEQ ID NO:7 is I or V, preferably V,    -   ii) X13 of SEQ ID NO:7 is G or S, preferably S,    -   iii) X72 of SEQ ID NO:7 is P, L or S, preferably L or S,        -   more preferably S,    -   iv) X98 of SEQ ID NO:7 is G or D, preferably D, and/or    -   v) X128 of SEQ ID NO:7 is G or C, preferably C.        That “at least one” of these conditions applies includes that,        e.g., one, two, three, four or all five conditions may apply. In        some embodiments of the invention, all five conditions apply,        i.e. the following applies for the inventive polypeptide        comprising an amino acid sequence exhibiting at least 80%        sequence identity with the sequence of SEQ ID NO:7:    -   i) X5 of SEQ ID NO:7 is I or V, preferably V,    -   ii) X13 of SEQ ID NO:7 is G or S, preferably S,    -   iii) X72 of SEQ ID NO:7 is P, L or S, preferably L or S,        -   more preferably S,    -   iv) X98 of SEQ ID NO:7 is G or D, preferably D, and    -   v) X128 of SEQ ID NO:7 is G or C, preferably C.

In some embodiments of the invention, at least one of the following fiveconditions applies for the inventive polypeptide comprising an aminoacid sequence exhibiting at least 80% sequence identity with thesequence of SEQ ID NO:7:

-   -   i) X5 of SEQ ID NO:7 is not I,    -   ii) X13 of SEQ ID NO:7 is not G,    -   iii) X72 of SEQ ID NO:7 is not P,    -   iv) X98 of SEQ ID NO:7 is not G, and/or    -   v) X128 of SEQ ID NO:7 is not G.        That “at least one” of these conditions applies includes that,        e.g., one, two, three, four or all five conditions may apply.

In some embodiments of the invention, at least one of the following fiveconditions applies for the inventive polypeptide comprising an aminoacid sequence exhibiting at least 80% sequence identity with thesequence of SEQ ID NO:7:

-   -   i) X5 of SEQ ID NO:7 is V,    -   ii) X13 of SEQ ID NO:7 is S,    -   iii) X72 of SEQ ID NO:7 is L or S, preferably S,    -   iv) X98 of SEQ ID NO:7 is D, and/or    -   v) X128 of SEQ ID NO:7 is C.        As before, that “at least one” of these conditions applies        includes that, e.g., one, two, three, four or all five        conditions may apply. Hence, in some embodiments of the        invention all five conditions apply, i.e. the following applies        for the inventive polypeptide comprising an amino acid sequence        exhibiting at least 80% sequence identity with the sequence of        SEQ ID NO:7:    -   i) X5 of SEQ ID NO:7 is V,    -   ii) X13 of SEQ ID NO:7 is S,    -   iii) X72 of SEQ ID NO:7 is L or S, preferably S,    -   iv) X98 of SEQ ID NO:7 is D, and    -   v) X128 of SEQ ID NO:7 is C.        If only one condition applies, it is particularly preferred if        this means that X72 of SEQ ID NO:7 is L or S, preferably S.        Aside of the situations, where only one of these condition        applies (i.e. X5 of SEQ ID NO:7 is V, X13 of SEQ ID NO:7 is S,        X72 of SEQ ID NO:7 is L or S, preferably S, X98 of SEQ ID NO:7        is D or X128 of SEQ ID NO:7 is C), particularly preferred        combinations are wherein at least i) X72 of SEQ ID NO:7 is L and        X98 of SEQ ID NO:7 is D, ii) X13 of SEQ ID NO:7 is S and X72 of        SEQ ID NO:7 is S, iii) X5 of SEQ ID NO:7 is V and X72 of SEQ ID        NO:7 is S, and/or iv) X72 of SEQ ID NO:7 is S and X128 of SEQ ID        NO:7 is C.

In particularly preferred embodiments of the invention, the polypeptidecomprising an amino acid sequence exhibiting at least 80% sequenceidentity with the sequence of SEQ ID NO:7 does again not exhibit theinactivating mutation E18A described in Oliveira et al. (PLoS One, 2014Oct. 7; 9(10):e108376) (see SEQ ID NO:4). The polypeptide comprising anamino acid sequence exhibiting at least 80% sequence identity with thesequence of SEQ ID NO:7 does thus preferably not comprise an alanineresidue at said position, i.e. does not comprise an amino acid sequencewhich deviates (inter alia) with a E12A mutation from SEQ ID NO:7. Morepreferably, the polypeptide comprising an amino acid sequence exhibitingat least 80% sequence identity with the sequence of SEQ ID NO:7 doesretain the original glutamate residue (E) at said position, i.e. retainsE12 of SEQ ID NO:7 and does not deviate from SEQ ID NO:7 at saidposition.

The amino acid sequence exhibiting at least 80% sequence identity withthe sequence of SEQ ID NO:7 may deviate from SEQ ID NO:7 for example atposition 29, i.e. the threonine residue may be replaced by any otheramino acid, e.g. an alanine residue. Other preferred regions in whichthe polypeptide may deviate from the sequence of SEQ ID NO:7 are forexample residues 127 to 142 of SEQ ID NO:7. Preferably, deviations fromSEQ ID NO:7 are due to conservative amino acid substitutions.

As mentioned above, the present invention relates to a polypeptidecomprising an amino acid sequence exhibiting at least about 80% sequenceidentity with the sequence of SEQ ID NO:7. In more preferred embodimentssaid amino acid sequence deviates less than 20% from SEQ ID NO:7. Forexample, the polypeptide according to the invention may comprise anamino acid sequence exhibiting at least about 85%, at least about 90%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, at least about 99%, at least about 99.5% or even 100%sequence identity with the sequence of SEQ ID NO:7. In cases where theinventive polypeptide comprises an amino acid sequence exhibiting 100%sequence identity with the sequence of SEQ ID NO:7, the inventivepolypeptide comprises the sequence of SEQ ID NO:7. Such embodiment isparticularly contemplated by the inventor.

In a further embodiment of the present invention, the polypeptidecomprising an amino acid sequence exhibiting at least about 90% sequenceidentity with the sequence of SEQ ID NO:1 is a polypeptide, whichcomprises an amino acid sequence exhibiting at least about 80% sequenceidentity with the sequence of SEQ ID NO:8, wherein SEQ ID NO:8 ischaracterized by

-   -   X5 of SEQ ID NO:8 may be any amino acid, preferably I or V,    -   X13 of SEQ ID NO:8 may be any amino acid, preferably G or S,    -   X72 of SEQ ID NO:8 may be any amino acid, preferably P, L or S,    -   X98 of SEQ ID NO:8 may be any amino acid, preferably G or D,    -   X128 of SEQ ID NO:8 may be any amino acid, preferably G or C,        and    -   X150 of SEQ ID NO:8 may be any amino acid, preferably A or V.        It is again understood that for such polypeptide still the        general proviso of the present application for polypeptides        applies, i.e. that such polypeptide does neither comprise the        sequence according to SEQ ID NO:2, nor the sequence according to        SEQ ID NO:3, nor the sequence according to SEQ ID NO:4.

In some embodiments of the invention, at least one of the following sixconditions applies for the inventive polypeptide comprising an aminoacid sequence exhibiting at least 80% sequence identity with thesequence of SEQ ID NO:8:

-   -   i) X5 of SEQ ID NO:8 is I or V, preferably V,    -   ii) X13 of SEQ ID NO:8 is G or S, preferably S,    -   iii) X72 of SEQ ID NO:8 is P, L or S, preferably L or S,        -   more preferably S,    -   iv) X98 of SEQ ID NO:8 is G or D, preferably D,    -   v) X128 of SEQ ID NO:8 is G or C, preferably C, and/or    -   vi) X150 of SEQ ID NO:8 is A or V, preferably V.        That “at least one” of these conditions applies includes that,        e.g., one, two, three, four, five or all six conditions may        apply. In some embodiments of the invention, all six conditions        apply, i.e. the following applies for the inventive polypeptide        comprising an amino acid sequence exhibiting at least 80%        sequence identity with the sequence of SEQ ID NO:8:    -   i) X5 of SEQ ID NO:8 is I or V, preferably V,    -   ii) X13 of SEQ ID NO:8 is G or S, preferably S,    -   iii) X72 of SEQ ID NO:8 is P, L or S, preferably L or S,        -   more preferably S,    -   iv) X98 of SEQ ID NO:8 is G or D, preferably D,    -   v) X128 of SEQ ID NO:8 is G or C, preferably C, and    -   vi) X150 of SEQ ID NO:8 is A or V, preferably V.

In some embodiments of the invention, at least one of the following sixconditions applies for the inventive polypeptide comprising an aminoacid sequence exhibiting at least 80% sequence identity with thesequence of SEQ ID NO:8:

-   -   i) X5 of SEQ ID NO:8 is not I,    -   ii) X13 of SEQ ID NO:8 is not G,    -   iii) X72 of SEQ ID NO:8 is not P,    -   iv) X98 of SEQ ID NO:8 is not G,    -   v) X128 of SEQ ID NO:8 is not G, and/or    -   vi) X150 of SEQ ID NO:8 is not A.        That “at least one” of these conditions applies includes that,        e.g., one, two, three, four, five or all six conditions may        apply.

In some embodiments of the invention, at least one of the following sixconditions applies for the inventive polypeptide comprising an aminoacid sequence exhibiting at least 80% sequence identity with thesequence of SEQ ID NO:8:

-   -   i) X5 of SEQ ID NO:8 is V,    -   ii) X13 of SEQ ID NO:8 is S,    -   iii) X72 of SEQ ID NO:8 is L or S, preferably S,    -   iv) X98 of SEQ ID NO:8 is D,    -   v) X128 of SEQ ID NO:8 is C, and/or    -   vi) X150 of SEQ ID NO:8 is V.        As before, that “at least one” of these conditions applies        includes that, e.g., one, two, three, four, five or all six        conditions may apply. Hence, in some embodiments of the        invention all six conditions apply, i.e. the following applies        for the inventive polypeptide comprising an amino acid sequence        exhibiting at least 80% sequence identity with the sequence of        SEQ ID NO:8:    -   i) X5 of SEQ ID NO:8 is V,    -   ii) X13 of SEQ ID NO:8 is S,    -   iii) X72 of SEQ ID NO:8 is L or S, preferably S,    -   iv) X98 of SEQ ID NO:8 is D,    -   v) X128 of SEQ ID NO:8 is C, and    -   vi) X150 of SEQ ID NO:8 is V.        If only one condition applies, it is particularly preferred if        this means that X72 of SEQ ID NO:8 is L or S, preferably S.        Aside of the situations, where only one of these condition        applies (i.e. X5 of SEQ ID NO:8 is V, X13 of SEQ ID NO:8 is S,        X72 of SEQ ID NO:8 is L or S, preferably S, X98 of SEQ ID NO:8        is D or X128 of SEQ ID NO:8 is C, X150 of SEQ ID NO:8 is V),        particularly preferred combinations are wherein at least i) X72        of SEQ ID NO:8 is L and X98 of SEQ ID NO:8 is D, ii) X13 of SEQ        ID NO:8 is S and X72 of SEQ ID NO:8 is S, iii) X5 of SEQ ID NO:8        is V and X72 of SEQ ID NO:8 is S, iv) X72 of SEQ ID NO:8 is S        and X128 of SEQ ID NO:8 is C, and/or v) X72 of SEQ ID NO:8 is L,        X98 of SEQ ID NO:8 is D and X150 of SEQ ID NO:8 is V.

In particularly preferred embodiments of the invention, the polypeptidecomprising an amino acid sequence exhibiting at least 80% sequenceidentity with the sequence of SEQ ID NO:8 does again not exhibit theinactivating mutation E18A described in Oliveira et al. (PLoS One, 2014Oct. 7; 9(10):e108376) (see SEQ ID NO:4). The polypeptide comprising anamino acid sequence exhibiting at least 80% sequence identity with thesequence of SEQ ID NO:8 does thus preferably not comprise an alanineresidue at said position, i.e. does not comprise an amino acid sequencewhich deviates (inter alia) with a E12A mutation from SEQ ID NO:8. Morepreferably, the polypeptide comprising an amino acid sequence exhibitingat least 80% sequence identity with the sequence of SEQ ID NO:8 doesretain the original glutamate residue (E) at said position, i.e. retainsE12 of SEQ ID NO:8 and does not deviate from SEQ ID NO:8 at saidposition.

The amino acid sequence exhibiting at least 80% sequence identity withthe sequence of SEQ ID NO:8 may deviate from SEQ ID NO:8 for example atposition 29, i.e. the threonine residue may be replaced by any otheramino acid, e.g. an alanine residue. Other preferred regions in whichthe polypeptide may deviate from the sequence of SEQ ID NO:8 are forexample residues 127 to 156 of SEQ ID NO:8. Preferably, deviations fromSEQ ID NO:8 are due to conservative amino acid substitutions.

As mentioned above, the present invention relates to a polypeptidecomprising an amino acid sequence exhibiting at least about 80% sequenceidentity with the sequence of SEQ ID NO:8. In more preferred embodimentssaid amino acid sequence deviates less than 20% from SEQ ID NO:8. Forexample, the polypeptide according to the invention may comprise anamino acid sequence exhibiting at least about 85%, at least about 90%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, at least about 99%, at least about 99.5% or even 100%sequence identity with the sequence of SEQ ID NO:8. In cases where theinventive polypeptide comprises an amino acid sequence exhibiting 100%sequence identity with the sequence of SEQ ID NO:8, the inventivepolypeptide comprises the sequence of SEQ ID NO:8. Such embodiment isparticularly contemplated by the inventor.

In a further embodiment of the present invention, the polypeptidecomprising an amino acid sequence exhibiting at least about 90% sequenceidentity with the sequence of SEQ ID NO:1 is a polypeptide, whichcomprises an amino acid sequence exhibiting at least about 80% sequenceidentity with the sequence of SEQ ID NO: 9, wherein SEQ ID NO: 9 ischaracterized by

-   -   X1 of SEQ ID NO: 9 may be absent or any amino acid, in        particular M,    -   X11 of SEQ ID NO: 9 may be any amino acid, preferably I or V,    -   X19 of SEQ ID NO: 9 may be any amino acid, preferably G or S,    -   X78 of SEQ ID NO: 9 may be any amino acid, preferably P, L or S,    -   X104 of SEQ ID NO: 9 may be any amino acid, preferably G or D,    -   X134 of SEQ ID NO: 9 may be any amino acid, preferably G or C,        and    -   X156 of SEQ ID NO: 9 may be any amino acid, preferably A or V.        It is again understood that for such polypeptide still the        general proviso of the present application for polypeptides        applies, i.e. that such polypeptide does neither comprise the        sequence according to SEQ ID NO:2, nor the sequence according to        SEQ ID NO:3, nor the sequence according to SEQ ID NO:4.

In some embodiments of the invention, at least one of the followingseven conditions applies for the inventive polypeptide comprising anamino acid sequence exhibiting at least 80% sequence identity with thesequence of SEQ ID NO: 9:

-   -   i) X1 of SEQ ID NO: 9 is absent or M, preferably M,    -   ii) X11 of SEQ ID NO: 9 is I or V, preferably V,    -   iii) X19 of SEQ ID NO: 9 is G or S, preferably S,    -   iv) X78 of SEQ ID NO: 9 is P, L or S, preferably L or S,        -   more preferably S,    -   v) X104 of SEQ ID NO: 9 is G or D, preferably D,    -   vi) X134 of SEQ ID NO: 9 is G or C, preferably C, and/or    -   vii) X156 of SEQ ID NO: 9 is A or V.        That “at least one” of these conditions applies includes that,        e.g., one, two, three, four, five, six or all seven conditions        may apply. In some embodiments of the invention, all seven        conditions apply, i.e. the following applies for the inventive        polypeptide comprising an amino acid sequence exhibiting at        least 80% sequence identity with the sequence of SEQ ID NO: 9:    -   i) X1 of SEQ ID NO: 9 is absent or M, preferably M,    -   ii) X11 of SEQ ID NO: 9 is I or V, preferably V,    -   iii) X19 of SEQ ID NO: 9 is G or S, preferably S,    -   iv) X78 of SEQ ID NO: 9 is P, L or S, preferably L or S,        -   more preferably S,    -   v) X104 of SEQ ID NO: 9 is G or D, preferably D,    -   vi) X134 of SEQ ID NO: 9 is G or C, preferably C, and    -   vii) X156 of SEQ ID NO: 9 is A or V.

In some embodiments of the invention, at least one of the followingseven conditions applies for the inventive polypeptide comprising anamino acid sequence exhibiting at least 80% sequence identity with thesequence of SEQ ID NO: 9:

-   -   i) X1 of SEQ ID NO: 9 is absent,    -   ii) X11 of SEQ ID NO: 9 is not I,    -   iii) X19 of SEQ ID NO: 9 is not G,    -   iv) X78 of SEQ ID NO: 9 is not P,    -   v) X104 of SEQ ID NO: 9 is not G,    -   vi) X134 of SEQ ID NO: 9 is not G, and/or    -   vii) X156 of SEQ ID NO: 9 is not A.        That “at least one” of these conditions applies includes that,        e.g., one, two, three, four, five, six or all seven conditions        may apply.

In some embodiments of the invention, at least one of the followingseven conditions applies for the inventive polypeptide comprising anamino acid sequence exhibiting at least 80% sequence identity with thesequence of SEQ ID NO: 9:

-   -   i) X1 of SEQ ID NO: 9 is M,    -   ii) X11 of SEQ ID NO: 9 is V,    -   iii) X19 of SEQ ID NO: 9 is S,    -   iv) X78 of SEQ ID NO: 9 is L or S, preferably S,    -   v) X104 of SEQ ID NO: 9 is D,    -   vi) X134 of SEQ ID NO: 9 is C, and/or    -   vii) X156 of SEQ ID NO: 9 is V.        As before, that “at least one” of these conditions applies        includes that, e.g., one, two, three, four, five, six or all        seven conditions may apply. Hence, in some embodiments of the        invention all seven conditions apply, i.e. the following applies        for the inventive polypeptide comprising an amino acid sequence        exhibiting at least 80% sequence identity with the sequence of        SEQ ID NO: 9:    -   i) X1 of SEQ ID NO: 9 is M,    -   ii) X11 of SEQ ID NO: 9 is V,    -   iii) X19 of SEQ ID NO: 9 is S,    -   iv) X78 of SEQ ID NO: 9 is L or S, preferably S,    -   v) X104 of SEQ ID NO: 9 is D,    -   vi) X134 of SEQ ID NO: 9 is C and    -   vii) X156 of SEQ ID NO: 9 is V.        If only one condition applies, it is particularly preferred if        this means that X78 of SEQ ID NO: 9 is L or S, preferably S.        Aside of the situations, where only one of these condition        applies (i.e. X1 of SEQ ID NO: 9 is M, X11 of SEQ ID NO: 9 is V,        X19 of SEQ ID NO: 9 is S, X78 of SEQ ID NO: 9 is L or S,        preferably S, X104 of SEQ ID NO: 9 is D, X134 of SEQ ID NO: 9 is        C, or X156 of SEQ ID NO: 9 is V), particularly preferred        combinations are wherein at least i) X78 of SEQ ID NO: 9 is L        and X104 of SEQ ID NO: 9 is D, ii) X19 of SEQ ID NO: 9 is S and        X78 of SEQ ID NO: 9 is S, iii) X11 of SEQ ID NO: 9 is V and X78        of SEQ ID NO: 9 is S, iv) X78 of SEQ ID NO: 9 is S and X134 of        SEQ ID NO: 9 is C, and/or v) X78 of SEQ ID NO: 9 is L, X104 of        SEQ ID NO: 9 is D and X156 of SEQ ID NO: 9 is V.

If the inventive polypeptide comprises one or more amino acid residuesN-terminal of the amino acid sequence exhibiting at least 80% sequenceidentity with the sequence of SEQ ID NO: 9, then it is preferred that X1of SEQ ID NO: 9 is not M, i.e. absent or any other amino acid. In thealternative constellation, i.e. where the inventive polypeptide does notcomprise one or more amino acid residues N-terminal of the amino acidsequence exhibiting at least 80% sequence identity with the sequence ofSEQ ID NO: 9, it is preferred if X1 of SEQ ID NO: 9 is M, in particularif the polypeptide is to be expressed by recombinant means.

In particularly preferred embodiments of the invention, the polypeptidecomprising an amino acid sequence exhibiting at least 80% sequenceidentity with the sequence of SEQ ID NO: 9 does again not exhibit theinactivating mutation E18A described in Oliveira et al. (PLoS One, 2014Oct. 7; 9(10):e108376) (see SEQ ID NO:4). The polypeptide comprising anamino acid sequence exhibiting at least 80% sequence identity with thesequence of SEQ ID NO: 9 does thus preferably not comprise an alanineresidue at said position, i.e. does not comprise an amino acid sequencewhich deviates (inter alia) with a E18A mutation from SEQ ID NO: 9. Morepreferably, the polypeptide comprising an amino acid sequence exhibitingat least 80% sequence identity with the sequence of SEQ ID NO: 9 doesretain the original glutamate residue (E) at said position, i.e. retainsE18 of SEQ ID NO: 9 and does not deviate from SEQ ID NO: 9 at saidposition.

The amino acid sequence exhibiting at least 80% sequence identity withthe sequence of SEQ ID NO: 9 may deviate from SEQ ID NO: 9 for exampleat position 35, i.e. the threonine residue may be replaced by any otheramino acid, e.g. an alanine residue. Other preferred regions in whichthe polypeptide may deviate from the sequence of SEQ ID NO: 9 are forexample residues 1 to 6 of SEQ ID NO: 9 and/or residues 133 to 162 ofSEQ ID NO: 9. Preferably, deviations from SEQ ID NO: 9 are due toconservative amino acid substitutions.

As mentioned above, the present invention relates to a polypeptidecomprising an amino acid sequence exhibiting at least about 80% sequenceidentity with the sequence of SEQ ID NO: 9. In more preferredembodiments said amino acid sequence deviates less than 20% from SEQ IDNO: 9. For example, the polypeptide according to the invention maycomprise an amino acid sequence exhibiting at least about 85%, at leastabout 90%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, at least about 99%, at least about 99.5% or even100% sequence identity with the sequence of SEQ ID NO: 9. In cases wherethe inventive polypeptide comprises an amino acid sequence exhibiting100% sequence identity with the sequence of SEQ ID NO: 9, the inventivepolypeptide comprises the sequence of SEQ ID NO: 9. Such embodiment isparticularly contemplated by the inventor.

The present invention relates to polypeptides exhibiting at least acertain level of sequence identity to a given fragment sequence of Lys68enzyme. Possible mutations, which do not negatively impact function ofthe enzyme, can be derived for example from the sequences of highlysimilar enzymes of Lys67 enzyme such as AFO12350.1 (SEQ ID NO:11),A0A0K1Y7J0 (SEQ ID NO:12), YP_224045.1 (SEQ ID NO:13), AFO12459.1 (SEQID NO:14), YP_009322827.1 (SEQ ID NO:15), WP_076927521.1 (SEQ ID NO:16),WP_076917165.1 (SEQ ID NO:17), WP_016047076.1 (SEQ ID NO:18), AFO70790.1(SEQ ID NO:19), WP_076915447.1 (SEQ ID NO:20), AGF87755.1 (SEQ IDNO:21), YP_009009975.1 (SEQ ID NO:22), YP_001110823.1 (SEQ ID NO:23),YP_008239773.1 (SEQ ID NO:24), YP_008767061.1 (SEQ ID NO:25),WP_034086662.1 (SEQ ID NO:26), APM00295.1 (SEQ ID NO:27), YP_007010505.1(SEQ ID NO:28), APU02985.1 (SEQ ID NO:29), YP_009280144.1 (SEQ IDNO:30), and YP_009035189.1 (SEQ ID NO:31).

In this context, the present invention does also relate a polypeptidecomprising an amino acid sequence exhibiting at least 90% sequenceidentity with the sequence of SEQ ID NO: 10, wherein SEQ ID NO: 10 ischaracterized by

-   -   X2 may be any amino acid, preferably D or N,    -   X5 may be any amino acid, preferably L, I or V,    -   X6 may be any amino acid, preferably H or K,    -   X13 may be any amino acid, preferably G, V or S,    -   X15 may be any amino acid, preferably R or Q,    -   X20 may be any amino acid, preferably K or R,    -   X23 may be any amino acid, preferably K or P,    -   X24 may be any amino acid, preferably S or N,    -   X28 may be any amino acid, preferably L or F,    -   X32 may be any amino acid, preferably Y or F,    -   X34 may be any amino acid, preferably H or S,    -   X37 may be any amino acid, preferably A or P,    -   X38 may be any amino acid, preferably D or H,    -   X40 may be any amino acid, preferably K or Y,    -   X49 may be any amino acid, preferably Q or R,    -   X55 may be any amino acid, preferably H or N,    -   X56 may be any amino acid, preferably K or R,    -   X59 may be any amino acid, preferably V, S or A,    -   X72 may be any amino acid, preferably P, L, T or S,    -   X81 may be any amino acid, preferably M or V,    -   X90 may be any amino acid, preferably V, P or A,    -   X95 may be any amino acid, preferably A or V,    -   X98 may be any amino acid, preferably G or D,    -   X108 may be any amino acid, preferably V, I or A,    -   X109 may be any amino acid, preferably A or S,    -   X113 may be any amino acid, preferably N or S,    -   X116 may be any amino acid, preferably S or T;        with the proviso that the polypeptide does neither comprise the        sequence according to SEQ ID NO:2, nor the sequence according to        SEQ ID NO:3, nor the sequence according to SEQ ID NO:4, nor        consists of any of the following sequences: SEQ ID NO:11, SEQ ID        NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,        SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID        NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,        SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID        NO:30, and SEQ ID NO:31. Preferably, the polypeptide comprises        the amino acid sequence according to SEQ ID NO: 10. It is also        preferred, that the polypeptide does not comprise any of the        following sequences: SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13,        SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID        NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,        SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID        NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID        NO:31. Preferably such polypeptide comprises S or L, preferably        S, at the position corresponding to X72.

Examples for inventive polypeptides are for instance polypeptidescomprising SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34, as always withthe proviso that such polypeptide does neither comprise the sequenceaccording to SEQ ID NO:2, nor the sequence according to SEQ ID NO:3, northe sequence according to SEQ ID NO:4. Other examples for inventivepolypeptides are for instance polypeptides comprising SEQ ID NO:35, SEQID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, or SEQ ID NO:40.

A polypeptide according to the present invention exhibits preferably theactivity of a peptidoglycan degrading enzyme, i.e. is capable ofdegrading bacterial peptidoglycan. Typically a polypeptide of thepresent invention will be capable of degrading the peptidoglycan ofbacteria of Gram-negative bacteria, such as Salmonella sp. bacteria. Thepeptidoglycan degrading activity on gram negative bacteria can bemeasured by assays well known in the art, e.g. by muralytic assays inwhich the outer membrane of gram negative bacteria is permeabilized orremoved (e.g. with chloroform) to allow the putative enzyme access tothe peptidoglycan layer. If the enzyme is active, degradation of thepeptidoglycan layer will lead to a drop of turbidity, which can bemeasured photometrically (see for example Briers et al., J. Biochem.Biophys Methods 70: 531-533, (2007).

A polypeptide according to the present invention may compriseadditionally at least one further amino acid sequence stretch selectedfrom the group consisting of amphipathic peptide, cationic peptide,polycationic peptide, hydrophobic peptide, or naturally occurringantimicrobial peptide, like sushi peptide and defensin. This additionalat least one amino acid sequence stretch may in principle be present atany position in the inventive polypeptide, but is preferably present atthe termini, i.e. in the N- or C-terminal region of the inventivepolypeptide. Such additional amino acid sequence stretch may be fuseddirectly, or via a peptide linker, to the rest of the polypeptide. It isunderstood that if one (or more) such additional amino acid sequencestretches according to the present invention are present in theN-terminal region of the inventive polypeptide, then there may befurther additional amino acids on the N-terminus of the additional aminoacid sequence stretch. Preferably these comprise the amino acidmethionine (Met), or the sequence methionine, glycine and serine(Met-Gly-Ser).

This at least one additional amino acid sequence stretch preferably hasthe function to lead the inventive polypeptide through the outermembrane of bacteria and may have activity or may have no or only lowactivity when administered without being fused to the polypeptide of theinvention. The function to guide the polypeptide through the outermembrane of Gram-negative bacteria is caused by the outer membrane orLPS disrupting, permeabilising or destabilizing activity of said aminoacid sequence stretches.

Such outer membrane or LPS disrupting or permeabilising or destabilizingactivity of these amino acid sequence stretches may be determined in amethod as follows: The bacteria cells to be treated are cultured inliquid medium or on agar plates. Then the bacteria cell concentration inthe liquid medium is determined photometrically at OD₆₀₀ nm or thecolonies on the agar plates are counted, respectively. Now, the bacteriacells in liquid medium or on the plates are treated with a polypeptideaccording to the present invention exhibiting at least one additionalamino acid sequence stretch as defined herein. After incubation thebacteria cell concentration in the liquid medium is determinedphotometrically at OD₆₀₀ nm or the colonies on the agar plates arecounted again. If the protein exhibits such outer membrane or LPSdisrupting or permeabilising or destabilizing activity, the bacteriacells are lysed due to the treatment with the polypeptide and thus, thebacteria cell concentration in the liquid medium or the number of thebacteria colonies on the agar plate is reduced. Thus, the reduction inbacteria cell concentration or in the number of bacteria colonies aftertreatment with the protein is indicative for an outer membrane or LPSdisrupting or permeabilising or destabilizing activity of thepolypeptide.

Especially preferred are cationic and/or polycationic amino acidsequence stretches comprising at least one motive according to SEQ IDNO:41 (KRKKRK). In particular cationic amino acid sequence stretchescomprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16or 17 motives according to SEQ ID NO: 41 (KRKKRK) are preferred. Morepreferred are cationic peptide stretches comprising at least one KRKmotive (lys-arg-lys), preferable at least 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32 or 33 KRK motives.

In another preferred embodiment of the present invention the cationicamino acid sequence stretch comprises beside the positively chargedamino acid residues, in particular lysine and/or arginine residues,neutrally charged amino acid residues, in particular glycine and/orserine residues. Preferred are cationic amino acid sequence stretchesconsisting of about 70% to about 100%, or about 80% to about 95%, orabout 85% to about 90% positively charged amino acid residues, inparticular lysine, arginine and/or histidine residues, more preferablylysine and/or arginine residues and of about 0% to about 30%, or about5% to about 20%, or about 10% to about 20% neutrally charged amino acidresidues, in particular glycine and/or serine residues. Preferred areamino acid sequence stretches consisting of about 4% to about 8% serineresidues, of about 33% to about 36% arginine residues and of about 56%to about 63% lysine residues. Especially preferred are amino acidsequence stretches comprising at least one motive according to SEQ IDNO: 42 (KRXKR), wherein X is any other amino acid than lysine, arginineand histidine. Especially preferred are polypeptide stretches comprisingat least one motive according to SEQ ID NO: 43 (KRSKR). More preferredare cationic stretches comprising at least about 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or at least about 20 motivesaccording to SEQ ID NO: 42 (KRXKR) or SEQ ID NO: 43 (KRSKR).

Also preferred are amino acid sequence stretches consisting of about 9to about 16% glycine residues, of about 4 to about 11% serine residues,of about 26 to about 32% arginine residues and of about 47 to about 55%lysine residues. Especially preferred are amino acid sequence stretchescomprising at least one motive according to SEQ ID NO: 44 (KRGSG). Morepreferred are cationic stretches comprising at least about 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or at least bout 20motives according to SEQ ID NO: 44 (KRGSG).

In another preferred embodiment of the present invention such cationicamino acid sequence stretch comprises beside the positively chargedamino acid residues, in particular lysine and/or arginine residues,hydrophobic amino acid residues, in particular valine, isoleucine,leucine, methionine, phenylalanine, tryptophan, cysteine, alanine,tyrosine, proline and glycine residues, more preferably alanine, valine,leucine, isoleucine, phenylalanine, and/or tryptophan residues.Preferred are cationic amino acid sequence stretches consisting of about70% to about 100%, or about 80% to about 95%, or about 85% to about 90%positively charged amino acid residues, in particular lysine and/orarginine residues and of about 0% to about 30%, or about 5% to about20%, or about 10% to about 20% hydrophobic amino acid residues, valine,isoleucine, leucine, methionine, phenylalanine, tryptophan, cysteine,alanine, tyrosine, proline and glycine residues, more preferablyalanine, valine, leucine, isoleucine, phenylalanine, and/or tryptophanresidues. Examples for cationic and polycationic amino acid sequencestretches are listed in the following table:

TABLE 1 amino acid sequence stretch length SEQ ID NO: KRKKRK 6 41KRKKRKKRK 9 45 RRRRRRRRR 9 46 KKKKKKKK 8 47 KRKKRKKRKK 10 48KRKKRKKRKKRK 12 49 KRKKRKKRKKRKKR 14 50 KKKKKKKKKKKKKKKK 16 51KRKKRKKRKKRKKRKKRK 18 52 KRKKRKKRKKRKKRKKRKK 19 53 RRRRRRRRRRRRRRRRRRR19 54 KKKKKKKKKKKKKKKKKKK 19 55 KRKKRKKRKRSKRKKRKKRK 20 56KRKKRKKRKRSKRKKRKKRKK 21 57 KRKKRKKRKKRKKRKKRKKRK 21 58KRKKRKKRKRGSGKRKKRKKRK 22 59 KRKKRKKRKRGSGSGKRKKRKKRK 24 60KRKKRKKRKKRKKRKKRKKRKKRKK 25 61 KRKKRKKRKRSKRKKRKKRKRSKRKKRKKRK 31 62KRKKRKKRKRGSGSGKRKKRKKRKGSGSGKRKKRKKRK 38 63KRKKRKKRKKRKKRKKRKKRKKRKKRKKRKKRKKRKKRK 39 64KRKKRKKRKRSKRKKRKKRKRSKRKKRKKRKRSKRKKRKKRK 42 65

In a further aspect of the present invention at least one of theadditional amino acid sequence stretches is an antimicrobial peptide,which comprises a positive net charge and around 50% hydrophobic aminoacids. The antimicrobial peptides are amphipathic with a length of about12 to about 50 amino acid residues. The antimicrobial peptides arenaturally occurring in insects, fish, plants, arachnids, vertebrates ormammals. Preferably the antimicrobial peptide may be naturally occurringin radish, silk moth, wolf spider, frog, preferably in Xenopus laevis,Rana frogs, more preferably in Rana catesbeiana, toad, preferably Asiantoad Bufo bufo gargarizans, fly, preferably in Drosophila, morepreferably in Drosophila melanogaster, in Aedes aegypti, in honey bee,bumblebee, preferably in Bombus pascuorum, flesh fly, preferably inSarcophaga peregrine, scorpion, horseshoe crab, catfish, preferably inParasilurus asotus, cow, pig, sheep, porcine, bovine, monkey and human.

In another preferred embodiment of the present invention theantimicrobial amino acid sequence stretches consist of about 0% to about5%, or about 0% to about 35%, or about 10% to about 35% or about 15% toabout 45%, or about 20% to about 45% positively charged amino acidresidues, in particular lysine and/or arginine residues and of about 50%to about 80%, or about 60% to about 80%, or about 55% to about 75%, orabout 70% to about 90% hydrophobic amino acid residues, valine,isoleucine, leucine, methionine, phenylalanine, tryptophan, cysteine,alanine, tyrosine, proline and glycine residues, more preferablyalanine, valine, leucine, isoleucine, phenylalanine, and/or tryptophanresidues.

In another preferred embodiment of the present invention theantimicrobial amino acid sequence stretches consist of about 4% to about58% positively charged amino acid residues, in particular lysine and/orarginine residues and of about 33% to about 89% hydrophobic amino acidresidues, valine, isoleucine, leucine, methionine, phenylalanine,tryptophan, cysteine, alanine, tyrosine, proline and glycine residues,more preferably alanine, valine, leucine, isoleucine, phenylalanine,and/or tryptophan residues.

Examples for antimicrobial amino acid sequences which may be used incarrying out the present invention are listed in the following table.

TABLE 2 Peptide Sequence SEQ ID NO LL-37LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPR 66 TES SMAP-29RGLRRLGRKIAHGVKKYGPTVLRIIRIAG 67 Indolicidin ILPWKWPWWPWRR 68 ProtegrinRGGRLCYCRRRFCVCVGR 69 Cecropin P1 SWLSKTAKKLENSAKKRISEGIAIAIQGGPR 70Magainin GIGKFLHSAKKFGKAFVGEIMNS 71 PleurocidinGWGSFFKKAAHVGKHVGKAALTHYL 72 Cecropin AGGLKKLGKKLEGAGKRVFNAAEKALPVVAGAKAL 73 (A. aegypti) RK Cecropin AGWLKKIGKKIERVGQHTRDATIQGLGIPQQAANV 74 (D. AATARG melanogaster)Buforin II TRSSRAGLQFPVGRVHRLLRK 75 Sarcotoxin IAGWLKKIGKKIERVGQHTRDATIQGLGIAQQAANV 76 AATAR Apidaecin ANRPVYIPPPRPPHPRL77 Ascaphine 5 GIKDWIKGAAKKLIKTVASHIANQ 78 Nigrocine 2GLLSKVLGVGKKVLCGVSGLVC 79 Pseudin 1 GLNTLKKVFQGLHEAIKLINNHVQ 80Ranalexin FLGGLIVPAMICAVTKKC 81 Melittin GIGAVLKVLTTGLPALISWIKRKRQQ 82Lycotoxin 1 IWLTALKFLGKHAAKKLAKQQLSKL 83 Parasin 1 KGRGKQGGKVRAKAKTRSS84 Buforin I AGRGKQGGKVRAKAKTRSSRAGLQFPVGRVHRLL 85 RKGNY Dermaseptin 1ALWKTMLKKLGTMALHAGKAALGAAADTISQGTQ 86 Bactenecin 1 RLCRIVVIRVCR 87Thanatin GSKKPVPITYCNRRTGKCQRM 88 Brevinin 1T VNPIILGVLPKVCLITKKC 89Ranateurin 1 SMLSVLKNLGKVGLGFVACKINIKQC 90 Esculentin 1GIFSKLGRKKIKNLLISGLKNVGKEVGMDVVRTG 91 IKIAGCKIKGEC TachyplesinRWCFRVCYRGICYRKCR 92 Androctonin RSVCRQIKICRRRGGCYYKCTNRPY 93 alpha-DCYCRIPACIAGERRYGTCIYQGRLWAFCC 94 defensin beta-NPVSCVRNKGICVPIRCPGSMKQIGTCVGRAVKC 95 defensin CRKK theta-GFCRCLCRRGVCRCICTR 96 defensin defensinATCDLLSGTGINHSACAAHCLLRGNRGGYCNGKA 97 (sapecin A) VCVCRN ThioninTTCCPSIVARSNFNVCRIPGTPEAICATYTGCII 98 (crambin) IPGATCPGDYANdefensin from QKLCQRPSGTWSGVCGNNNACKNQCIRLEKARHG 99 radishSCNYVFPAHCICYFPC Drosomycin DCLSGRYKGPCAVWDNETCRRVCKEEGRSSGHCS 100PSLKCWCEGC Hepcidin DTHFPICIFCCGCCHRSKCGMCCKT 101 Bac 5RFRPPIRRPPIRPPFYPPFRPPIRPPIFPPIRPP 102 FRPPLGRPFP PR-39RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRF 103 PPRFP PyrrhocoricinVDKGSYLPRPTPPRPIYNRN 104 Histatin 5 DSHAKRHHGYKRKFHEKHHSHRGY 105 ECP19RPPQFTRAQWFAIQHISLN 106 MSI-594 GIGKFLKKAKKGIGAVLKVLTTG 107 TL-ColMMETLTVHAPSPSTNLPSYGNGAFSLSAPHVPGAG 108 P SBO KLKKIAQKIKNFFAKLVA 109

In a further aspect of the present invention at least one of theadditional amino acid sequence stretches may be a sushi peptide which isdescribed by Ding J L, Li P, Ho B Cell Mol Life Sci. 2008 April;65(7-8):1202-19. The Sushi peptides: structural characterization andmode of action against Gram-negative bacteria. Especially preferred isthe sushi 1 peptide according to SEQ ID NO: 110.

Preferred sushi peptides are sushi peptides S1 and S3 and multiplesthereof; FASEB J. 2000 September; 14(12):1801-13.

In a further aspect of the present invention at least one of theadditional amino acid sequence stretches is a hydrophobic peptide, whichcomprises at least 90% of the hydrophobic amino acid residues of valine,isoleucine, leucine, methionine, phenylalanine, tryptophan, cysteine,alanine, tyrosine, proline and/or glycine. In another preferredembodiment the hydrophobic peptide fused to the protein of the inventionconsists of about 90% to about 95%, or of about 90% to about 100%, or ofabout 95% to about 100% of the hydrophobic amino acid residues ofvaline, isoleucine, leucine, methionine, phenylalanine, tryptophan,cysteine, alanine, tyrosine, proline and/or glycine.

Preferred hydrophobic peptides are Walmagh1 having the amino acidsequence according to SEQ ID NO: 111 and the hydrophobic peptide havingthe amino acid sequence Phe-Phe-Val-Ala-Pro (SEQ ID NO: 112).

In a further aspect of the present invention at least one of theadditional amino acid sequence stretches is an amphipathic peptide,which comprises one or more of the positively charged amino acidresidues of lysine, arginine and/or histidine, combined to one or moreof the hydrophobic amino acid residues of valine, isoleucine, leucine,methionine, phenylalanine, tryptophan, cysteine, alanine, tyrosine,proline and/or glycine. Side chains of the amino acid residues areoriented in order that cationic and hydrophobic surfaces are clusteredat opposite sides of the peptide. Preferably, more than about 30, 40,50, 60 or 70% of the amino acids in said peptide are positively chargedamino acids. Preferably, more than about 30, 40, 50, 60 or 70%, of theamino acid residues in said peptide are hydrophobic amino acid residues.Advantageously, the amphipathic peptide is present at the N-terminal orthe C-terminal end of the polypeptide according to the presentinvention.

In another embodiment of the invention, the amphipathic peptide consistsof at least 5, more preferably at least of 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49or at least 50 amino acid residues. In a preferred embodiment at leastabout 30, 40, 50, 60 or 70% of said amino acid residues of theamphipathic peptide are either arginine or lysine residues and/or atleast about 30, 40, 50, 60 or 70% of said amino acid residues of theamphipathic peptide are of the hydrophobic amino acids valine,isoleucine, leucine, methionine, phenylalanine, tryptophan, cysteine,alanine, tyrosine, proline and/or glycine.

In another preferred embodiment of the present invention the amphipathicpeptide stretch comprises beside the positively charged amino acidresidues, in particular lysine and/or arginine residues, hydrophobicamino acid residues, in particular valine, isoleucine, leucine,methionine, phenylalanine, tryptophan, cysteine, alanine, tyrosine,proline and glycine residues, more preferably alanine, valine, leucine,isoleucine, phenylalanine, and/or tryptophan residues. Preferred areamphipathic peptide stretches consisting of about 10% to about 50%, orabout 20% to about 50%, or about 30% to about 45% or about 5% to about30% positively charged amino acid residues, in particular lysine and/orarginine residues and of about 50% to about 85%, or about 50% to about90%, or about 55% to about 90%, or about 60% to about 90%, or about 65%to about 90% hydrophobic amino acid residues, valine, isoleucine,leucine, methionine, phenylalanine, tryptophan, cysteine, alanine,tyrosine, proline and glycine residues, more preferably alanine, valine,leucine, isoleucine, phenylalanine, and/or tryptophan residues. Inanother preferred embodiment amphipathic peptide stretches consisting of12% to about 50% positively charged amino acid residues, in particularlysine and/or arginine residues and of about 50% to about 85%hydrophobic amino acid residues, valine, isoleucine, leucine,methionine, phenylalanine, tryptophan, cysteine, alanine, tyrosine,proline and glycine residues, more preferably alanine, valine, leucine,isoleucine, phenylalanine, and/or tryptophan residues.

Preferred amphipathic peptides are α4-helix of T4 lysozyme according toSEQ ID NO: 113 and WLBU2-Variant having the amino acid sequenceaccording to SEQ ID NO: 114 and Walmagh 2 according to SEQ ID NO: 115.

The optional additional amino acid sequence stretches as specified aboveconsist preferably of at least 5, more preferably at least of 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99 or at least 100 amino acid residues. Especially preferred are thoseadditional amino acid sequence stretches consisting of about 5 to about100 amino acid residues, about 5 to about 50 or about 5 to about 30amino acid residues. More preferred are peptide stretches consisting ofabout 6 to about 42 amino acid residues, about 6 to about 39 amino acidresidues, about 6 to about 38 amino acid residues, about 6 to about 31amino acid residues, about 6 to about 25 amino acid residues, about 6 toabout 24 amino acid residues, about 6 to about 22 amino acid residues,about 6 to about 21 amino acid residues, about 6 to about 20 amino acidresidues, about 6 to about 19 amino acid residues, about 6 to about 16amino acid residues, about 6 to about 14 amino acid residues, about 6 toabout 12 amino acid residues, about 6 to about 10 amino acid residues orabout 6 to about 9 amino acid residues.

In a preferred embodiment the inventive polypeptide comprises at leastone amino acid sequence stretch selected from the group consisting ofKRK and SEQ ID NOs: 41-115. Preferably, the inventive polypeptidecomprises at least one amino acid sequence stretch selected from thegroup consisting of KRK and SEQ ID NOs: 41-115 (see in particular tables1 and 2), and an amino acid sequence selected from any one of SEQ ID NO:1 and SEQ ID NO: 5 to 40, wherein preferably the amino acid sequencestretches, are fused to the N- and/or C-terminus of the amino acidsequence selected from the group consisting of SEQ ID NO: 1 and SEQ IDNO: 5 to 40.

The additional amino acid sequence stretch of the polypeptide accordingto the present invention may be linked to the rest of the enzyme byintervening additional amino acid residues e.g. due to cloning reasons.Alternatively, the additional amino acid sequence stretches may bedirectly linked to the rest of the enzyme sequence without interveninglinker sequences. The additional amino acid sequences, if more than onepresent in the inventive polypeptide and positioned on the same terminusof the enzyme, may likewise be linked to each other by additionalintervening amino acid residues or may be directly joined to each other.

Preferably, said intervening additional amino acid residues may not berecognized and/or cleaved by proteases. Preferably said additional aminoacid sequences are linked to each other and/or to the enzyme by at least1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional intervening amino acidresidues.

In a preferred embodiment the at least one additional amino acidsequence stretch is linked to the rest of the inventive polypeptide,preferably at the N- or C-terminus of the polypeptide according to thepresent invention, by the additional intervening amino acid residuesglycine, serine and serine (Gly-Ser-Ser), glycine, alanine, glycine andalanine (Gly-Ala-Gly-Ala; SEQ ID NO:116), glycine, alanine, glycine,alanine, glycine, alanine, glycine and alanine(Gly-Ala-Gly-Ala-Gly-Ala-Gly-Ala; SEQ ID NO:117) or glycine, alanine,glycine, alanine, glycine, alanine, glycine, alanine, glycine, alanine,glycine and alanine (Gly-Ala-Gly-Ala-Gly-Ala-Gly-Ala-Gly-Ala-Gly-Ala;SEQ ID NO:118).

Aside of the enzymatic domain (i.e. a domain having the activity ofdegrading the peptidoglycan of Gram-negative bacteria, such as SEQ IDNO:1), and the optional additional amino acid sequence stretches, asdefined herein, the inventive polypeptide may of course also compriseother amino acid sequence elements, e.g. one or more tags, e.g. aHis-tag, Strep-tag, Avi-tag, Myc-tag, Gst-tag, JS-tag, cystein-tag,FLAG-tag or other tags known in the art, thioredoxin, maltose bindingproteins (MBP) etc.

In this context, the inventive polypeptide, preferably having theability of degrading the peptidoglycan layer of Gram negative bacteriasuch as Salmonella bacteria, may additional comprise a tag e.g. forpurification. Preferred is a His₆-tag (SEQ ID NO: 119), preferably atthe C-terminus and/or the N-terminus of the polypeptide according to thepresent invention. Said tag can be linked to the polypeptide byadditional amino acid residues e.g. due to cloning reasons. Preferablysaid tag can be linked to the protein by at least 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 additional amino acid residues. Preferably said additionalamino acid residues may not be recognized and/or cleaved by proteases.In a preferred embodiment the inventive polypeptide comprises a His₆-tagat its C-terminus linked to the polypeptide by the additional amino acidresidues lysine and glycine (Lys-Gly) or leucine and glutamic acid(Leu-Glu). Preferably, said additional amino acid residues may be notrecognized or cleaved by proteases. In another preferred embodiment theinventive polypeptide comprises a His₆-tag at its N-terminus linked tothe polypeptide by the additional amino acid residues lysine and glycine(Lys-Gly) or leucine and glutamic acid (Leu-Glu). In another preferredembodiment the polypeptide comprises a His₆-tag at its N- and C-terminuslinked to the polypeptide by the additional amino acid residues lysineand glycine (Lys-Gly) or leucine and glutamic acid (Leu-Glu).

A polypeptide according to the present invention can be produced bystandard means known in the art, e.g. by recombinant expression ofnucleic acids encoding the respective polypeptide in appropriate hostcells. If the inventive polypeptide comprises for example additionallyamino acid sequence stretches or tags etc., such fusion proteins may beproduced by linking the required individual nucleic acid sequences usingstandard cloning techniques as described e.g. by Sambrook et al. 2001,Molecular Cloning: A Laboratory Manual. Such a polypeptide may beproduced likewise with methods known in the art, e.g., in recombinantDNA expression systems.

III. Nucleic Acids, Vectors, Bacteriophages and Host Cells

The present invention does also relate to nucleic acids encoding one ormore inventive polypeptide of the present invention. The inventivenucleic acid may take all forms conceivable for a nucleic acid. Inparticular the nucleic acids according to the present invention may beRNA, DNA or hybrids thereof. They may be single-stranded ordouble-stranded. The may have the size of small transcripts or of entiregenomes, such as a bacteriophage genome. As used herein, a nucleic acidencoding one or more inventive polypeptides of the present invention maybe a nucleic acid reflecting the sense strand. Likewise, the antisensestrand is also encompassed. The nucleic acid may encompass aheterologous promotor for expression of the inventive polypeptide.

In a further aspect the present invention relates to a vector comprisinga nucleic acid according to the present invention. Such vector may forexample be an expression vector allowing for expression of an inventivepolypeptide. Said expression may be constitutive or inducible. Thevector may also be a cloning vector comprising the nucleic acid sequenceof the current invention for cloning purposes.

The present invention does also relate to a bacteriophage comprising aninventive nucleic acid.

The present invention does also relate to (isolated) host cellscomprising a polypeptide, nucleic acid, vector, or bacteriophageaccording to the present invention. The host cells may be selected inparticular from the group consisting of bacterial cells and yeast cells.Where appropriate, other suitable host cells may be immortalized celllines, e.g. of mammalian (in particular human) origin.

IV. Compositions

In a further aspect the present invention relates to a compositioncomprising a polypeptide according to the present invention, a nucleicacid according to the present invention, a vector according to thepresent invention, a bacteriophage according to the present inventionand/or a host cell according to the present invention.

A composition according to the present invention may be a pharmaceuticalcomposition comprising a pharmaceutical acceptable diluent, excipient orcarrier.

BRIEF DESCRIPTION OF THE FIGURES

In the following a brief description of the appended FIGURE will begiven. The FIGURE is intended to illustrate the present invention inmore detail. However, it is not intended to limit the scope of theinvention to these specific examples.

FIG. 1 : illustrates the muralytic activity of polypeptides according toLys68(1-132) (SEQ ID NO:32), Lys68(1-148) (SEQ ID NO:33) andLys68(7-162) (SEQ ID NO:34). Buffer served as control. X-Axis: minutes.Y-Axis: OD₆₀₀.

V. Examples

In the following, specific examples illustrating various embodiments andaspects of the invention are presented. However, the present inventionshall not to be limited in scope by the specific embodiments describedherein. Indeed, various modifications of the invention in addition tothose described herein will become readily apparent to those skilled inthe art from the foregoing description, accompanying FIGURE and theexamples below. All such modifications fall within the scope of theappended claims.

Example 1: Truncated Lys68 Endolysin does Still Exhibit Activity

Lys68 is a globular endolysin, i.e. does not exhibit an apparent domainstructure with an enzymatic domain and a cell wall binding domain, asencountered for various other endolysins. The inventor hypothesized,that Lys68 endolysin may nonetheless exhibit a core region responsiblefor enzymatic activity and tested this hypothesis with truncatedversions of Lys68, namely Lys68(1-132) (SEQ ID NO:32), Lys68(1-148) (SEQID NO:33) and Lys68(7-162) (SEQ ID NO:34).

Briefly, the following experiment was carried out: Exponentially growingP. aeruginosa cells were harvested by centrifugation and subsequentlyresuspended in 0.05 M Tris/HCl pH 7.7 buffer saturated with chloroform.This cell suspension was incubated for 45 minutes at room temperature.Afterwards, cells were washed with 20 mM HEPES pH 7.4 and finallyadjusted to an OD600 of ca. 1.5 with 20 mM HEPES pH 7.4. In order totest the muralytic activity, 270 μl of chloroform treated cells weremixed with 30 μl of purified variants of Lys68 in a 96 well plate andthe OD600 was monitored in a microplate reader.

The result is shown in FIG. 1 . All constructs showed activity.

Example 2: Identification of Mutations Stabilizing Lys68 Endolysin

The inventor tested whether a polypeptide comprising SEQ ID NO:120 andfurther sequence elements would tolerate mutations in said sequence ofSEQ ID NO:120 (i.e. not leading to a loss of function), which ideallyexhibit in addition a positive effect on polypeptide stability.

Briefly, the following experiment was carried out: The purified variantsof Lys68 were incubated in 20 mM HEPES pH 7.4 and 500 mM NaCl at giventemperatures for 20 min. Subsequently, the protein solutions were cooleddown to 4° C. and the minimal inhibitory concentration (MIC) wasdetermined using Salmonella Manhattan (RKI 13-05699) cells. Therefore,exponentially growing cells with an OD600 of 0.6 are diluted 1:10 withMueller-Hinton medium (not cation-adjusted). This bacterial solution isthen further diluted 1:500 in Mueller-Hinton medium (notcation-adjusted). 180 μl of bacterial suspension are mixed with 18 μl ofprotein solution (20 mM HEPES pH 7.4, 500 mM NaCl) with increasingprotein concentration in a 96 well plate. Additionally, EDTA is added toa final concentration of 500 μM. The 96 well plate is incubated for 18to 20 hours at 37° C. The bacterial growth is subsequently determined ina microplate reader using a wavelength of OD600. The lowest proteinconcentration at which no bacterial growth is observed, is defined asthe minimal inhibitory concentration (MIC).

Some of the most promising candidates identified are illustrated in thefollowing table.

TABLE 3 Mutation RT 37° C. 39.6° C. 42.2° C. — 10 >45 >45 >45 P78L 20 1010 >45 P78L 10 10 10 >45 G104D A156V P78S 10 10 10 10 G19S 10 10 10 20P78S I11V 10 10 10 20 P78S P78S 15 15 15 25 G134C

Table 3 indicates the minimal inhibitory concentration (MIC; μg/ml) forthe various polypeptides which have been tested. “>” is intended toindicate, that the respective polypeptide did not show any inhibitoryactivity up to the indicated concentration. Concentrations above saidvalue have not been tested. The position of the mutation is indicatedwith respect to the position in full length Lys68 (SEQ ID NO:2). Thecorresponding polypeptides comprise the modified Lys68 sequencesaccording to SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQID NO:39, and SEQ ID NO:40, respectively. “- - - ” represents theunmutated control.

Example 3: Preferred Embodiments of the Invention

In the following, particularly preferred embodiments of the inventionare provided in items 1 to 31 below. Any aspect and particularembodiment discussed above with regard to the present invention may alsobe implemented in the context of the embodiments below:

-   -   1. Polypeptide comprising an amino acid sequence exhibiting at        least 90% sequence identity with the sequence of SEQ ID NO:1,        wherein SEQ ID NO:1 is characterized by        -   X5 may be any amino acid, preferably I or V,        -   X13 may be any amino acid, preferably G or S,        -   X72 may be any amino acid, preferably P, L or S,        -   X98 may be any amino acid, preferably G or D;        -   with the proviso that the polypeptide does neither comprise            the sequence according to SEQ ID NO:2, nor the sequence            according to SEQ ID NO:3, nor the sequence according to SEQ            ID NO:4.    -   2. The polypeptide according to item 1, wherein the polypeptide        comprises an amino acid sequence exhibiting at least about 80%        sequence identity with the sequence of SEQ ID NO: 9, wherein SEQ        ID NO: 9 is characterized by        -   X1 may be absent or any amino acid, in particular M,        -   X11 may be any amino acid, preferably I or V,        -   X19 may be any amino acid, preferably G or S,        -   X78 may be any amino acid, preferably P, L or S,        -   X104 may be any amino acid, preferably G or D,        -   X134 may be any amino acid, preferably G or C; and        -   X156 may be any amino acid, preferably A or V.    -   3. Polypeptide according to item 2, wherein at least one of the        following applies:        -   X1 of SEQ ID NO: 9 is absent or M,        -   X11 of SEQ ID NO: 9 is I or V,        -   X19 of SEQ ID NO: 9 is G or S,        -   X78 of SEQ ID NO: 9 is P, L or S,        -   X104 of SEQ ID NO: 9 is G or D,        -   X134 of SEQ ID NO: 9 is G or C, and/or        -   X156 of SEQ ID NO: 9 is A or V.    -   4. Polypeptide according to item 2 or item 3, wherein the        following applies:        -   X1 of SEQ ID NO: 9 is absent or M,        -   X11 of SEQ ID NO: 9 is I or V,        -   X19 of SEQ ID NO: 9 is G or S,        -   X78 of SEQ ID NO: 9 is P, L or S,        -   X104 of SEQ ID NO: 9 is G or D,        -   X134 of SEQ ID NO: 9 is G or C, and        -   X156 of SEQ ID NO: 9 is A or V.    -   5. Polypeptide according to any one of items 2 to 4, wherein at        least one of the following applies:        -   X1 of SEQ ID NO: 9 is M,        -   X11 of SEQ ID NO: 9 is V,        -   X19 of SEQ ID NO: 9 is S,        -   X78 of SEQ ID NO: 9 is L or S,        -   X104 of SEQ ID NO: 9 is D,        -   X134 of SEQ ID NO: 9 is C and/or        -   X156 of SEQ ID NO: 9 is V.    -   6. The polypeptide according to any one of items 2 to 5, wherein        the polypeptide exhibits in the amino acid sequence exhibiting        at least about 80% sequence identity with the sequence of SEQ ID        NO: 9 a glutamate residue (E) at the position corresponding to        position 18 of SEQ ID NO: 9.    -   7. The polypeptide according to any one of items 2 to 6, wherein        the amino acid sequence exhibiting at least 80% sequence        identity with the sequence of SEQ ID NO: 9 exhibits at least        85%, at least 90%, at least 95%, at least 96%, at least 97%, at        least 98%, at least 99%, at least 99.5% or even 100% sequence        identity with the sequence of SEQ ID NO: 9.    -   8. The polypeptide according to any one of items 2 to 7, wherein        the polypeptide comprises the sequence of SEQ ID NO: 9.    -   9. The polypeptide according to any one of items 2 to 8, wherein        X11 of SEQ ID NO: 9 is V.    -   10. The polypeptide according to any one of items 2 to 9,        wherein X19 of SEQ ID NO: 9 is S.    -   11. The polypeptide according to any one of items 2 to 10,        wherein X104 of SEQ ID NO: 9 is D.    -   12. The polypeptide according to any one of items 2 to 11,        wherein X134 of SEQ ID NO: 9 is C.    -   13. The polypeptide according to any one of items 2 to 12,        wherein X156 of SEQ ID NO: 9 is V.    -   14. The polypeptide according to any one of items 2 to 13,        wherein X78 of SEQ ID NO: 9 is L.    -   15. The polypeptide according to item 14, wherein X104 of SEQ ID        NO: 9 is D.    -   16. The polypeptide according to item 15, wherein X156 of SEQ ID        NO: 9 is V.    -   17. The polypeptide according to any one of items 2 to 13,        wherein X78 of SEQ ID NO: 9 is S.    -   18. The polypeptide according to item 17, wherein X19 of SEQ ID        NO: 9 is S.    -   19. The polypeptide according to item 17 or 18, wherein X11 of        SEQ ID NO: 9 is V.    -   20. The polypeptide according to any one of items 17 to 19,        wherein X134 of SEQ ID NO: 9 is C.    -   21. The polypeptide according to any one of items 2 to 20,        wherein X1 of SEQ ID NO: 9 is not M.    -   22. The polypeptide according to item 1, wherein the polypeptide        comprises a sequence selected from the group consisting of SEQ        ID NO: SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35,        SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ        ID NO:40.    -   23. The polypeptide according to any one of the preceding items,        wherein the polypeptide is capable of degrading the        peptidoglycan of Salmonella bacteria.    -   24. The polypeptide according to any one of the preceding items,        wherein the polypeptide comprises additionally at least one        amino acid sequence stretch selected from the group consisting        of amphiphatic peptide, cationic peptide, polycationic peptide,        hydrophobic peptide, naturally occurring antimicrobial peptide,        sushi peptide and defensin, wherein said amino acid sequence        stretch is preferably at the N-terminus.    -   25. The polypeptide according to any one of the preceding items,        wherein the polypeptide comprises at least one additional amino        acid sequence stretch selected from the group consisting of: KRK        and SEQ ID NOs: 41-115.    -   26. The polypeptide according to any one of the preceding items,        wherein the polypeptide has an overall length not exceeding 300        amino acids.    -   27. Nucleic acid encoding a polypeptide according to any one of        items 1 to 26.    -   28. Vector comprising a nucleic acid according to item 27.    -   29. Host cell comprising a polypeptide according to any one of        items 1 to 26, a nucleic acid according to item 27, and/or a        vector according to item 28.    -   30. Composition comprising a polypeptide according to any one of        items 1 to 26, a nucleic acid according to item 27, a vector        according to item 28 and/or a host cell according to item 29.    -   31. Composition according to item 30, wherein the composition is        a pharmaceutical composition comprising a pharmaceutical        acceptable diluent, excipient or carrier.

The invention claimed is:
 1. A nucleic acid encoding a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, wherein SEQ ID NO:1is characterized by: X5 may be any amino acid, X13 may be any aminoacid, X72 may be L or S, X98 may be any amino acid; with the provisothat the polypeptide does neither comprise the sequence according to SEQID NO:2, nor the sequence according to SEQ ID NO:3, nor the sequenceaccording to SEQ ID NO:4.
 2. The nucleic acid according to claim 1,wherein the encoded polypeptide comprises the amino acid sequence of SEQID NO: 9, wherein SEQ ID NO: 9 is characterized by: X1 may be absent orany amino acid, X11 may be any amino acid, X19 may be any amino acid,X78 may be L or S, X104 may be any amino acid, X134 may be any aminoacid, and X156 may be any amino acid.
 3. The nucleic acid according toclaim 2, wherein at least one of the following applies: X1 of SEQ ID NO:9 is absent or M, X11 of SEQ ID NO: 9 is I or V, X19 of SEQ ID NO: 9 isG or S, X104 of SEQ ID NO: 9 is G or D, X134 of SEQ ID NO: 9 is G or C,and/or X156 of SEQ ID NO: 9 is A or V.
 4. The nucleic acid according toclaim 2, wherein the following applies: X1 of SEQ ID NO: 9 is absent orM, X11 of SEQ ID NO: 9 is I or V, X19 of SEQ ID NO: 9 is G or S, X104 ofSEQ ID NO: 9 is G or D, X134 of SEQ ID NO: 9 is G or C, and X156 of SEQID NO: 9 is A or V.
 5. The nucleic acid according to claim 2, wherein atleast one of the following applies: X1 of SEQ ID NO: 9 is M, X11 of SEQID NO: 9 is V, X19 of SEQ ID NO: 9 is S, X104 of SEQ ID NO: 9 is D, X134of SEQ ID NO: 9 is C and/or X156 of SEQ ID NO: 9 is V.
 6. The nucleicacid according to claim 2, wherein X1 of SEQ ID NO: 9 is not M.
 7. Thenucleic acid according to claim 1, wherein the encoded polypeptidecomprises a sequence selected from the group consisting of SEQ ID NO:35,SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ IDNO:40.
 8. The nucleic acid according to claim 2, wherein the encodedpolypeptide is capable of degrading the peptidoglycan of Salmonellabacteria.
 9. The nucleic acid according to claim 2, wherein the encodedpolypeptide comprises additionally at least one amino acid sequencestretch selected from the group consisting of amphipathic peptide,cationic peptide, polycationic peptide, hydrophobic peptide, naturallyoccurring antimicrobial peptide, sushi peptide and defensin.
 10. Thenucleic acid according to claim 2, wherein the encoded polypeptidecomprises at least one additional amino acid sequence stretch selectedfrom the group consisting of: KRK and SEQ ID NOs: 41-115.
 11. Thenucleic acid according to claim 2, wherein the encoded polypeptide hasan overall length not exceeding 300 amino acids.
 12. A compositioncomprising a nucleic acid according to claim
 1. 13. A compositionaccording to claim 12, wherein the composition is a pharmaceuticalcomposition comprising a pharmaceutical acceptable diluent, excipient orcarrier.
 14. The nucleic acid according to claim 9, wherein the encodedamino acid sequence stretch is at the N-terminus.